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. 2024 Mar 26;27:126–169. doi: 10.1016/j.reth.2024.03.008

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells

Zailing Yang a,b,1, Chuan Tian a,1, Zhixu He c, Xiangqing Zhu a, Jie He a, Hang Pan a, Ye Li a, Guangping Ruan a, XiJun Wu b,⁎⁎, Xinghua Pan a,
PMCID: PMC10988135  PMID: 38571892

Abstract

Background

A decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear.

Methods

Aged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes.

Results

MSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth.

Conclusion

MSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

Keywords: Thymus aging, MSCs, TECs, DNA methylation

1. Background

The thymus is the main lymphoid organ and is composed of thymic cells and thymic stromal cells. Thymic stromal cells include thymic epithelial cells (TECs), fibroblasts, dendritic cells and endothelial cells [1,2]. TECs are composed of cortical TECs (cTECs) and medullary TECs (mTECs). They maintain T-cell development at different stages through positive and negative selection, so TECs are an indispensable component of T lymphocyte maturation in the thymus [3,4]. However, in humans and mice, the thymus begins to degenerate after birth, and the reticular structure of the thymus epithelium is gradually replaced by adipose tissue, accompanied by a decrease in T-cell output [5,6]. Some experiments have shown that TECs have lower proliferation potential and that there is a higher percentage of apoptotic and senescent TECs in elderly animals, indicating that these cells may be a potential target for thymus rejuvenation therapy [[7], [8], [9]]. Therefore, the decrease in the number and activity of TECs may be the key factors related to the unfavorable changes in thymus structure and function in ageing.

The best way to reverse thymus ageing is to stimulate growth of the thymus, but this is a challenging scientific and technical problem involving developmental biology. Mesenchymal stem cells (MSCs) have multidirectional differentiation potential and immune regulation capability, secreting a variety of bioactive factors and playing important roles in inflammation, tissue damage repair and aging degeneration. In a study of a rhesus monkey aging model [[10], [11], [12], [13]], we found that bone marrow MSCs (BMMSCs) derived from infant rhesus monkeys restored the structure and function of aging ovaries by promoting follicular and vascular regeneration, reducing the degree of ovarian fibrosis, and inhibiting apoptosis; improved lung structure and function by reducing fibrosis and inhibiting apoptosis, enhancing the division and proliferation of alveolar type II epithelial cells and improving their antioxidative stress ability; and significantly reversed the expression of age-related proteins in serum. In our previous study, it was also observed that MSCs could improve the structure and function of the thymus in elderly mice [14], but this observation needed to be confirmed with further studies in nonhuman primates, which are more closely related to humans. Moreover, the related cellular and epigenetic molecular regulatory mechanisms have not been clarified, leaving this an unanswered scientific question in the field.

DNA methylation is an epigenetic modification mode that regulates gene transcription without changing gene structure. It can turn gene transcription on or off through demethylation or methylation modification of specific genes and their promoters [15,16]. Previous studies have found that the high stability of DNA methylation is an important mechanism for stabilizing gene transcription, which regulates cell growth, division, proliferation, differentiation and secretory function by regulating gene opening and closing [17]. The methylation level of the functional gene itself is also closely related to the transcriptional expression of the gene, but whether it inhibits or promotes gene transcription is also related to the methylation site.

Therefore, we treated elderly macaques with MSCs and explored whether MSCs promote thymus tissue regeneration by acting on TECs in vivo; cocultured senescent TECs with MSCs to reveal the changes in genomic methylation and transcriptional profiles; and clarified the molecular regulatory mechanism to provide theoretical support for the use of MSCs in the treatment of thymic senescence.

2. Results

2.1. MSCs improving tissue structure and function of the thymus in elderly macaque monkeys

Elderly macaque monkeys aged 22–24 years old were treated with MSCs, which was injected into the femoral vein at a dose of 1 × 107/kg once every other day for a total of 3 times. The gross anatomy, morphology, tissue structure and function of the thymus were observed 5 months after the last treatment. In terms of thymus morphology and tissue structure, the results showed that the thymus tissue of elderly macaque monkeys obviously atrophied and degenerated and was replaced by a large amount of adipose tissue; the boundary of the cortical-medullary junction (CMJ) was blurred, and the number of thymocytes decreased. After MSC treatment, the thymus tissue regenerated, revealing that the volume of the thymus increased, the number of thymic epithelial cells, thymocytes and Hassall bodies increased, and the amount of adipose tissue decreased (Fig. 1A and B). In terms of thymus function, the thymus index and the levels of thymosin α and thymopoietin in peripheral blood were decreased, and the decreasing trend was reversed after MSCs treatment (Fig. 1C and D, Table 1). Finally, we detected the expression level of the aging marker gene P16 and the TEC marker molecule CK14 [18], and the results showed that P16 expression decreased, and the proportion of CK14 cells expressing the TEC marker molecule increased after MSCs treatment, (Fig. 1E and F). Those results implied that MSCs treatment improving the structure and function of aging thymus and promoting the regeneration of thymic lobules.

Fig. 1.

Fig. 1

MSCs treatment changed thymus morphology, tissue structure, secretory function, and P16 and CK14 expression in aged macaque monkeys. A Comparison of the appearance and morphology of thymus between the Elderly and the Treated: the above figure shows the appearance of thymus of the aged macaque monkeys, represents the area of adipose tissue in the thymus. The figure on the below shows the appearance of the thymus after MSCs treatment, and represents the thymus tissue area. B HE staining of thymus tissue from the senescent elderly group and treatment group (100×, Image 1represents thymus cortex, Image 2represents the thymus medulla; Image 3 represents the Hassel body). C and D Comparison of thymus index and the contents of thymopoietin α and thymosin in the elderly and treatment group. E Expression of P16 in the elderly and treatment group (400×). F The expression of CK14 in thymus tissue was detected by immunohistochemistry (400×, brown indicates antibody-positive reaction) (the measured values represent the mean ± standard deviation (`X ± s) of three replicate experiments. ∗∗P < 0.01, ∗∗∗P < 0.001).

Table 1.

Thymus index.

Group Thymus massx(g) Macaque body weight (kg) Thymus index (organ weight g per kg body weight) Convert to percentage
Elderly1 2 5.3 0.377358490566038 37.7358490566038
Elderly2 2.8 5 0.56 56
Elderly3 3 5.6 0.535714285714286 53.5714285714286
Treated1 5 6 0.833333333333333 83.3333333333333
Treated2 5.2 5.8 0.896551724137931 89.6551724137931
Treated3 4.9 5.3 0.924528301886793 92.4528301886793

2.2. Intervention effect of MSCs on senescent TECs in vitro

To demonstrate that MSCs promote thymus tissue regeneration by promoting the growth and differentiation of TECs, we induced the senescence of TECs with 200 μmol/L H2O2 in vitro and cocultured MSCs with ageing TECs for 48 h in Transwells. Firstly, SA-β-gal (senescence–associated β-galactosidase, senescent cells appear blue due to increased β-galactosidase activity) staining showed that 55.3 ± 5.03% of the TECs in the induced senescence group were stained blue, and the number of blue cells decreased significantly after coculture (31.33 ± 7.03%) (Fig. 2A). Secondly, Dynamic observation by a Nanolive 4D microscope showed that MSCs secreted vesicles into the ageing TECs during coculture, which changed the volume of the TECs from large to small, and the loose nucleus became compact (Supplementary video 1). Then, under transmission electron microscopy, heterochromatin aggregation and patchy distribution were observed in senescent TECs, mitochondria were swollen, and no obvious autophagosomes were found in the cells. After coculture, the chromatin distribution of TECs was uniform, the mitochondria were still swollen, and a small number of autophagosomes could be seen in the cells (Fig. 2B). Next, western bloting and immunocytochemical staining were performed to detect the expression of the P16, P21 and Tunel protein, and the results showed that the expression of the ageing-related genes of P16, P21, and apoptosis increased significantly in the induced senescence group (1.59 ± 0.5, 1.17 ± 0.1, 85.66 ± 3.5%), and the expression of P16, P21 and Tunel was decreased after co-cultured with MSCs (1.14 ± 0.3, 1.10 ± 0.3, 33.06 ± 4.3%) (Fig. 2C and D). These results suggested that MSCs improve morphological structure and inhibit the expression of the aging markers SA-β-gal, P16, and P21, and decrease apoptosis to restore the growth of senescent TECs.

Fig. 2.

Fig. 2

Intervention effect of MSCs on aging TECs. A SA-β-gal staining of TECs after MSCs coculture (100 ×, represents increased activity of SA-β-gal). B Ultrastructural changes in TECs after MSC coculture (nucleus (N), mitochondria (M), and autophagosome (ASS); the upper scale is 2 μm). C After coculture with MSCs, the TEC senescence genes P16 and P21 were expressed. D TUNEL staining was used to detect the effect of MSCs on H2O2-induced apoptosis (the measured values represent the mean ± standard deviation (`X ± s) of three replicate experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Supplementary video related to this article can be found at https://doi.org/10.1016/j.reth.2024.03.008

The following is/are the supplementary data related to this article:

Video S1

3

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2.3. Changes in the genomic methylation profile in senescent TECs after MSCs coculture

To explore the intervention effect of MSCs on the whole-genome methylation modification profile of senescent TECs, we used whole-genome methylation sequencing (WGBS) to analyze the whole-genome methylation modification map of TECs after aging and MSCs coculture (Fig. 3A). The bioinformatics analysis method was used to conduct a series analysis of the WGBS database, resulting in 76.1 billion WGBS paired terminal readings (Table 2). The average alignment rate of clean WGBS reads mapped to the reference genome (BSMAP (version: 2.90) was 90.8%, and the coverage rate of the reference C base was 95%. We found that the average genome-wide methylation level of TECs in the induced senescence group was 64.13%. After coculture, the average methylation level of TECs increased (Fig. 3B). In the site methylation level analysis, 57.58% of the CG sites in the induced senescence group were hypermethylated (methylation levels ranged from 70 to 100%). The proportion of hypermethylated CG sites increased after MSCs coculture, while the proportion of hypomethylated CG sites (methylation level <30%) decreased after MSCs coculture (Fig. 3C). Subsequently, we analyzed the methylation level around the gene body, and the methylation level decreased gradually in the upstream 1 kb region of the gene. A significant decrease in methylation level was observed closer to the gene transcription start site (TSS). The methylation level in the gene body region was higher than that in the adjacent region and was relatively stable, while the methylation level in the transcription termination site (TTS) decreased significantly (Fig. 3D). Furthermore, the methylation changes in the TEC gene regulatory series after MSCs coculture were determined, and the annotations of senescence-related genes were extracted from the Gene Ontology Consortium (http://wiki.geneontology.org/index.php)). It was found that 1092 genes had methylation differences in the promoter region (Table 3). Among them, the methylation level in the promoter region of 501 genes was increased, and 23 genes were mainly involved in negative regulation of growth, proliferation and promotion of apoptosis. The methylation level of 591 genes decreased in the promoter region; 37 of these genes promoted cell growth, proliferation and anti-apoptosis (Fig. 3E and F) (Table 4). These results suggest that MSCs methylate the promoter region of genes related to apoptosis promotion, cell cycle arrest and growth inhibition in senescent TECs and turn off the expression of genes related to growth promotion, cell proliferation and apoptosis inhibition.

Fig. 3.

Fig. 3

Changes in the genomic methylation profile of senescent TECs after MSC coculture. A DNA methylation analysis workflow. B The average methylation level of the whole genome of senescent TECs after MSCs coculture. C The methylation level of CG loci in senescent TECs after MSCs coculture (orange lines indicate the percentage of low-methylated CG loci, blue histogram shows the percentage of hypermethylated CG loci). D DNA methylation levels at different locations of the gene (abscissa for different locations of the gene, ordinate for methylation level). E The differentially methylated genes in senescent TECs after MSCs coculture (red is the increase in methylation level, green is the decrease in demethylation level). F Enrichment analysis of ageing-related differentially methylated genes in TECs after MSCs coculture (the abscissa is the number of genes, and the ordinate is the GO name).

Table 2.

Overview of WGBS and RNA-seq.

Sample Basic data
Mapping_summary
Cytosines covgerage summary
Before filter Clean Data(bp) After filter Clean Data(bp) After filter Q20(%) After filter GC(%) BS Conversion Rate(%) Mapped Reads Mapped Ratio(%) Cover_C_site Coverage_C_rate(%) Cover_CG_site Coverage_CG_rate(%) Cover_CHG_site Coverage_CHG_rate(%) Cover_CHH_site Coverage_CHH_rate(%)
induced senescence1 129935007000.00 128996537171.00 96.44 21.14 99.1726 785756297 91.08 1139204604 94.99 54112170 92.2 241600401 95.26 843492031 95.1
induced senescence2 123296597700.00 122388023155.00 96.14 21.38 99.1842 743637765 90.84 1148446802 95.76 55009497 93.73 243620231 96.06 849817072 95.82
induced senescence3 121363978800.00 120054543421.00 96.38 21.38 99.153 723202127 90.06 1151420689 96.01 55225916 94.1 244218586 96.29 851976185 96.06
co-culture1 135175752300.00 134069766573.00 96.46 21.34 99.1876 816867744 91.01 1150752262 95.96 55194530 94.04 244111610 96.25 851446120 96
co-culture2 123507014100.00 122701110877.00 96.41 21.18 99.1815 748481642 91.23 1154482488 96.27 55464297 94.5 244859659 96.54 854158530 96.3
co-culture3 128602854900.00 127696268168.00 96.63 20.92 99.177 782219000 91.52 1142153850 95.24 54414238 92.71 242234242 95.51 845505368 95.33
Mean 126980200800.00 125984374894.17 96.41 21.2233333333333 99.1759833333334 766694095.833333 90.9566666666667 1147743449.16667 95.705 54903441.3333333 93.5466666666667 243440788.166667 95.985 849399217.666667 95.7683333333333
Total 761881204800.00 755906249365.00 4600164575
Sample Basic data
Mapping summary
sequenced_Total_Genes
RawDatas CleanData AF_Q20(%) AF_Q30(%) Total_Mapped Total_Mapped(%) Unique_Mapped Unique_Mapped(%) Multiple_Mapped Multiple_Mapped(%)
induced senescence1 63242212 63106352 98.17 94.45 61526591 97.77 59881074 95.16 1645517 2.61 14728 72.57
induced senescence2 88342632 88112024 98.23 94.32 86149774 98.06 83834209 95.42 2315565 2.64 14879 73.31
induced senescence3 72322932 72143332 98.04 94.19 70243386 97.68 68278367 94.95 1965019 2.73 14829 73.07
co-culture1 68212998 68069126 98.09 94.33 66420866 97.84 64655493 95.24 1765373 2.6 14709 72.48
co-culture2 61494366 61382532 98.13 94.36 59913926 97.88 58267348 95.19 1646578 2.69 14661 72.24
co-culture3 61247622 61114060 98.06 94.24 59525003 97.66 57956113 95.09 1568890 2.57 14616 72.02
Mean 69143793.6666667 68987904.3333333 98.12 94.315 67296591 97.815 65478767.3333333 95.175 1817823.66666667 2.64 14737 72.615
Total 414862762 413927426 403779546 88422

Table 3.

Differential methylation analysis.

id meth.diff meth.q
ENSG00000004534 11.1410214995872 0.0429895835444528
ENSG00000004534 13.2101994115724 0.0147077633344553
ENSG00000004660 12.8033744321869 0.0449216378457375
ENSG00000005206 11.9883161229276 0.00486888246659608
ENSG00000005206 16.4079172001707 0.000413089603869239
ENSG00000006611 32.9668348954578 0.0274261593096852
ENSG00000007171 19.2361927144536 0.0284955000718223
ENSG00000007264 21.036621036621 0.0247241716820028
ENSG00000007264 35.1750456280073 0.00800363463509878
ENSG00000010244 11.3078264865938 0.0127295348224089
ENSG00000011105 13.3912721893491 0.0370655291928072
ENSG00000025796 13.0936251519889 0.0253294671351397
ENSG00000025796 24.7747747747748 0.000261255054250883
ENSG00000027075 23.8587326822621 0.00152721677265883
ENSG00000027075 23.8587326822621 0.00152721677265883
ENSG00000028277 12.8211382113821 0.0417754562654187
ENSG00000035499 19.3525068415346 0.0442726305485908
ENSG00000048991 10.4895380349152 0.00234708565200044
ENSG00000056487 19.3288934426229 0.000346324136728837
ENSG00000057935 11.1359937892835 0.0216673574540229
ENSG00000064300 15.9053070905649 0.0313626607273396
ENSG00000065325 30.0664243639554 0.028033745947325
ENSG00000067225 14.5976635003821 0.000140304077436467
ENSG00000070087 12.0041443068912 0.000751864169014396
ENSG00000073146 28.3368254463589 1.79227087841047E-11
ENSG00000073670 13.6111807121579 0.0281038318353025
ENSG00000073849 15.5321329234373 0.0344241141481933
ENSG00000073861 14.2395496716356 0.0290369177541354
ENSG00000074211 31.0123456790123 0.0268393437770578
ENSG00000074219 11.9744703965236 0.00251060480696872
ENSG00000078124 21.7836257309941 0.0447363519040377
ENSG00000078549 24.8746457379551 3.10311426032942E-07
ENSG00000078549 19.6142930856553 3.5156097137167E-06
ENSG00000080371 14.5424394206038 0.00141882564824424
ENSG00000080845 12.6346909919374 0.029591386415805
ENSG00000083067 14.2926613616269 0.000291709269699351
ENSG00000083444 14.6650035637919 0.0276533853657112
ENSG00000083444 23.5729847494553 0.0147972826294937
ENSG00000083454 31.5746753246753 0.00664142162914082
ENSG00000088992 29.6296296296296 6.35666270161635E-07
ENSG00000088992 26.0344238002964 2.45996608894593E-08
ENSG00000088992 15.3504296759858 0.00698771447860245
ENSG00000089157 13.5674931129477 0.0398751044579701
ENSG00000089199 10.3386382673208 0.0247314804971351
ENSG00000089693 20.4041378101166 0.0169419692816553
ENSG00000091127 10.4413102820746 0.0474615788486051
ENSG00000091844 12.4604847207587 0.00656893745208257
ENSG00000092330 13.1012070566388 0.000153119337328558
ENSG00000092841 13.6121856866538 0.00589854211004375
ENSG00000095596 13.8338208211461 0.000133266843577669
ENSG00000095906 18.4410777507012 0.00106578761632672
ENSG00000096080 13.1624423963134 3.60710679412085E-13
ENSG00000099385 15.1779124333683 0.000505029650335907
ENSG00000099995 27.3128175630333 3.96812611779901E-05
ENSG00000100014 14.238574492658 0.00257404896789635
ENSG00000100038 36.9134363159429 3.7826902450755E-05
ENSG00000100147 18.4098639455782 0.0182070272285959
ENSG00000100209 15.8402203856749 0.000754006150568186
ENSG00000100288 28.4500466853408 0.00675645469505879
ENSG00000100344 33.1310942578548 0.0349828784568553
ENSG00000100739 23.1958939264328 0.00642102513264005
ENSG00000101057 11.4852240425329 0.027582108815626
ENSG00000101152 10.0759013282732 0.0183802377303916
ENSG00000101152 15.6436312692794 5.99465610526218E-08
ENSG00000101557 14.5645645645646 0.00498789412500717
ENSG00000101557 20.9821428571429 0.00296388715080506
ENSG00000101608 21.7574085059191 0.0119172703899002
ENSG00000101751 15.6150627615063 0.00847306283066711
ENSG00000102195 28.8316266577136 4.86516419633658E-05
ENSG00000102539 19.0187693038727 0.0241727783256494
ENSG00000103056 14.531033834334 0.0271381137967538
ENSG00000103089 23.6152005888848 4.1037077680861E-06
ENSG00000103197 13.6897001303781 0.0385416787972231
ENSG00000103260 34.8668280871671 0.00576313236691994
ENSG00000104142 15.6246783986827 0.0355032569018309
ENSG00000104765 15.1990920268609 0.0215482067013419
ENSG00000104853 10.1353178509232 0.0182451782141719
ENSG00000105186 16.5007301208018 0.00452513955835274
ENSG00000105186 16.5007301208018 0.00452513955835274
ENSG00000106013 18.0428023785807 0.000524037914938989
ENSG00000106034 10.4788838612368 0.0288752690171604
ENSG00000106038 13.2367119708892 0.0116835243402002
ENSG00000106462 18.861272695223 0.000787825751585364
ENSG00000106689 12.1085577087978 0.0439548340409723
ENSG00000107551 10.7314782200305 0.0353103544330602
ENSG00000107831 16.358024691358 0.0329967000955916
ENSG00000107957 27.9484536082474 0.00724694443189657
ENSG00000108100 15 0.0336594557786585
ENSG00000108518 13.0415847497698 0.0355546305919269
ENSG00000108599 11.4219114219114 0.00649490868695054
ENSG00000108641 14.9391044088868 0.00674109795438447
ENSG00000108848 15.4737757171834 0.0352960887961454
ENSG00000108950 15.8198128545928 0.000286189101485006
ENSG00000109158 20 0.0374828243738455
ENSG00000109390 14.8784878487849 8.36294203163539E-05
ENSG00000109390 10.5591956919649 0.000460100120298561
ENSG00000109906 23.1024365352724 0.0024547054883833
ENSG00000109906 20.4666732955487 0.00140854131257951
ENSG00000110628 12.6218323586745 0.000905019819790039
ENSG00000110768 22.6938199415264 0.0266440194266571
ENSG00000110768 22.6938199415264 0.0266440194266571
ENSG00000111057 19.963963963964 0.000494478725805716
ENSG00000111057 16.6082144805549 0.0189635681399841
ENSG00000111271 14.5110267534346 0.0359526622537845
ENSG00000112312 14.2666860408796 0.000252933316932294
ENSG00000112312 12.0671851302075 0.00314034552387615
ENSG00000112539 19.8119889253751 0.0293719445323917
ENSG00000112837 15.3775922785966 0.00225950731419919
ENSG00000114026 16.5888093013599 0.000262572797788145
ENSG00000114026 12.4730430727983 0.0483647303353267
ENSG00000114405 17.9489421227487 0.0239681627595053
ENSG00000114978 14.891975308642 0.0277631947392702
ENSG00000114982 11 0.0237828902907722
ENSG00000115053 18.3673469387755 0.0345977207069055
ENSG00000115053 13.5922330097087 0.00228971892261384
ENSG00000115183 16.325731840605 0.00978959984261563
ENSG00000115507 14.2919726839325 2.65335702645699E-05
ENSG00000115507 16.5384896605359 7.80832598349621E-08
ENSG00000115596 19.7204968944099 0.0471322320159808
ENSG00000115596 18.5969387755102 0.0267926070508599
ENSG00000115648 12.9906661552231 0.000432032159732779
ENSG00000115648 11.4707653079293 0.00146399547053104
ENSG00000115657 20.1540832049307 0.0090588160999464
ENSG00000115839 15.513830002019 0.0282685367705523
ENSG00000115946 11.0898606987897 0.0151098790541535
ENSG00000116273 11.4664169119615 0.0291279908353443
ENSG00000117151 12.0481071754796 0.038042836527265
ENSG00000117597 23.6773179675223 0.00375105384414285
ENSG00000119004 16.7674418604651 0.0493690355960005
ENSG00000119547 13.0022197558269 0.0110827481784365
ENSG00000119547 15.0617079629279 0.00388583376741845
ENSG00000119547 24.4766505636071 0.000287897780947743
ENSG00000119599 11.9913818624387 0.0438215878070369
ENSG00000119953 12.8063228783438 0.0214469380195495
ENSG00000120063 27.1062271062271 0.000537666955164323
ENSG00000120063 28.0362443368224 1.64865091843957E-09
ENSG00000120063 24.7522457258766 0.00277354571261337
ENSG00000120088 25.1940719830628 0.00169770212477983
ENSG00000120215 11.0236220472441 0.006057045539393
ENSG00000120438 17.6411657559199 0.0197989462066386
ENSG00000121005 17.2958735733099 0.031629656826347
ENSG00000121210 20.8273208273208 0.0159666152087347
ENSG00000121236 17.0111238971998 2.8674824329256E-05
ENSG00000121236 17.4084595959596 0.000242040623929651
ENSG00000121314 21.285580231467 0.0498568351160869
ENSG00000121314 21.285580231467 0.0498568351160869
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ENSG00000178234 -18.9923469387755 0.0193669089939285
ENSG00000178234 -22.2923816060398 9.30459330102401E-05
ENSG00000178645 -12.648988053376 0.0469137584786117
ENSG00000179094 -13.4310850439883 0.0355759656096261
ENSG00000179331 -21.9194342095502 0.000235854083096956
ENSG00000180287 -24.0886330665918 0.00852227254789977
ENSG00000180818 -11.8653524270039 0.0285551186534443
ENSG00000180834 -18.060221475675 0.0201026712084086
ENSG00000181104 -13.6891228909896 0.0435232730481889
ENSG00000181104 -18.0640976638058 1.14813876834968E-05
ENSG00000181104 -22.6280986455405 0.000111828657367155
ENSG00000181192 -12.6157900070944 0.00754488631590946
ENSG00000181350 -18.2209259934378 0.000396760300992857
ENSG00000181392 -13.9805515702216 0.000969860854511591
ENSG00000182111 -10.9364529819075 0.012680610291506
ENSG00000182149 -35.0862068965517 0.00826722019021418
ENSG00000182256 -16.1525907792738 0.038574221482599
ENSG00000182348 -18.1128025756191 0.0194939041437827
ENSG00000182508 -13.0498281786942 0.032867434303606
ENSG00000182508 -14.4618262692722 0.00166153295956751
ENSG00000182687 -26.2898150453897 0.000434071699893402
ENSG00000183048 -18.5163996948894 0.0208771553597651
ENSG00000183715 -10.8016877637131 0.00612124554796258
ENSG00000183733 -10.0493848806289 0.00256565663957455
ENSG00000183837 -30.594441063096 0.00594580436285597
ENSG00000183837 -28.5073037127206 5.99242522804126E-05
ENSG00000184209 -19.0350877192982 0.00357549061671981
ENSG00000184209 -14.5669093648004 0.0101738819058995
ENSG00000184220 -10.7100415923945 0.0189969303520165
ENSG00000184226 -20.1388888888889 0.00122241909616555
ENSG00000184254 -14.297763338748 0.0098100477552559
ENSG00000184445 -28.9085545722714 1.10468522189305E-19
ENSG00000184470 -10.5525364848336 1.22641272400171E-07
ENSG00000184486 -11.9779353821907 0.0197742056775144
ENSG00000184486 -14.6947506993582 0.0308993694062091
ENSG00000184903 -17.3484848484848 0.0100846794029084
ENSG00000185085 -12.7741935483871 0.00355548903022132
ENSG00000185164 -28.9317134478425 0.00766039313216227
ENSG00000185198 -20.9677419354839 0.00800363463509878
ENSG00000185274 -36.7777777777778 4.13866236096393E-08
ENSG00000185551 -25 4.12989508384209E-05
ENSG00000186094 -15.7203751065644 0.0214515366994642
ENSG00000186994 -17.9708162334701 0.0380436486008334
ENSG00000187049 -21.4250497288291 0.000190407930326322
ENSG00000187094 -30.3257394084732 0.0148136929111902
ENSG00000187773 -24.3055555555556 0.010424771548779
ENSG00000187800 -18.8722605235133 0.00097347513700514
ENSG00000188051 -13.5283993115318 0.0284862998415288
ENSG00000188051 -16.4764492753623 0.00830673370301929
ENSG00000188177 -24.021164021164 0.00403947268078688
ENSG00000188389 -18.1108669779365 0.00597294639909556
ENSG00000188501 -16.9491525423729 0.0264267102325099
ENSG00000188529 -15.3846153846154 0.0488977801585413
ENSG00000188554 -16.2792058516196 2.78883070497941E-05
ENSG00000188706 -10.5804820799856 0.0407427459034703
ENSG00000188706 -18.876314577561 0.000205941242025698
ENSG00000188807 -14.1644210192597 0.00214158534118136
ENSG00000188921 -16.1585066317341 0.0076260149012931
ENSG00000188921 -17.3934430588423 0.00829600867835282
ENSG00000188981 -10.4969898808761 0.0245604522604636
ENSG00000189099 -10.7433380084151 0.0471893075050864
ENSG00000189120 -26.8765133171913 0.0118293720940106
ENSG00000189120 -16.2003903708523 0.0123035857744294
ENSG00000189134 -21.6028708133971 0.0420091386650367
ENSG00000196110 -13.8728706833698 0.0420544804809331
ENSG00000196123 -11.8119658119658 0.0011790794908365
ENSG00000196381 -10.4694508425264 0.0102714888088208
ENSG00000196407 -16.2735824568326 1.63439555606411E-10
ENSG00000196407 -16.9854280510018 3.65742313994721E-10
ENSG00000196440 -21.7175066312997 0.0141480507556097
ENSG00000196460 -12.2420683467906 9.84035291008814E-05
ENSG00000196636 -15.222285848148 0.0128524806273957
ENSG00000196653 -28.7365177195686 0.000498526638073357
ENSG00000196923 -10.1846285959639 0.0125269750370792
ENSG00000197079 -15.5820105820106 0.0385511399220718
ENSG00000197081 -21.8181818181818 0.00318594967466865
ENSG00000197128 -17.6767676767677 0.0134216004209188
ENSG00000197128 -20.2912102591504 0.0247558735443318
ENSG00000197451 -11.3869559539578 0.00256448161697349
ENSG00000197451 -21.9268510258698 1.00569142011319E-07
ENSG00000197557 -28.8865979381443 0.0260989117867014
ENSG00000197766 -27.8711484593837 0.0367699367462675
ENSG00000197785 -15.5625079271128 0.0058251957156053
ENSG00000197935 -21.5945242456377 0.000214694174478014
ENSG00000197991 -15.5700753252682 0.0450051741900873
ENSG00000198125 -10.6796116504854 0.041804252368166
ENSG00000198125 -10.6796116504854 0.041804252368166
ENSG00000198171 -11.3691260430391 0.0492812514169258
ENSG00000198208 -12.0056651051313 0.0134097078395219
ENSG00000198231 -10.7800751879699 0.0425875740986927
ENSG00000198464 -16.8132902878666 0.0230462164545817
ENSG00000198561 -10.5533596837945 0.00500520808218314
ENSG00000198633 -12.5153938057164 0.0405828566192806
ENSG00000198729 -15.178272046496 0.00629617981725982
ENSG00000198797 -14.8354404976921 0.00662519298755446
ENSG00000198885 -24.6820349761526 0.025466515731314
ENSG00000198911 -10.7121944078466 0.00210231479703907
ENSG00000198944 -15.3587625526401 0.0293127616163585
ENSG00000203883 -12.3390634262004 0.0146275199407374
ENSG00000204128 -24.5085483767689 0.0052252260379734
ENSG00000204128 -33.2793334876186 9.50251948882142E-06
ENSG00000204161 -18.4558131926553 0.0110080534024279
ENSG00000204161 -16.8376205787781 0.0451923472289257
ENSG00000204308 -18.1073374959397 0.0218284313380497
ENSG00000204308 -19.8579431772709 0.0166281458453145
ENSG00000204397 -25.547055684042 0.036555069792132
ENSG00000204599 -22.2016522344979 0.00142926075835363
ENSG00000204599 -15.4369398207426 0.0188931632771714
ENSG00000204704 -14.9842086793777 0.0295866997565394
ENSG00000204704 -14.9842086793777 0.0295866997565394
ENSG00000204889 -13.903743315508 4.01874758669504E-05
ENSG00000204909 -10.4985439596305 0.000245160528169384
ENSG00000204920 -10.3541162486425 0.0375594462005039
ENSG00000205221 -10.2626190688151 0.0166542415969332
ENSG00000205364 -14.3932579717191 0.0209663026917388
ENSG00000205364 -22.7849311575727 2.75613743411553E-08
ENSG00000205544 -25.2622377622378 0.0374908539061022
ENSG00000205659 -20.3245942571785 0.00640464663479342
ENSG00000205659 -17.2039116262224 0.00192938109327571
ENSG00000205702 -31.1950970377937 0.00353438535321241
ENSG00000205702 -30.6146572104019 0.00836764608199518
ENSG00000205730 -16.9912609238452 0.00548780640878567
ENSG00000206172 -17.1837108213224 0.00285232058509603
ENSG00000206262 -12.5686982199984 0.0372534055870424
ENSG00000206262 -13.4922856787358 0.0125498653646383
ENSG00000211644 -10.7890102148644 0.00621622387146682
ENSG00000211647 -22.3450830713965 0.0119776508408652
ENSG00000212747 -17.5645161290323 0.034797418404917
ENSG00000213463 -25.126213592233 0.0333275081725526
ENSG00000213999 -18.0312897671981 7.66402129046302E-11
ENSG00000213999 -11.0689307272696 2.42044046543343E-05
ENSG00000214212 -20.3813785733369 0.00225671200629606
ENSG00000214891 -13.9580680757151 0.0388767095822126
ENSG00000215217 -25.9943714821764 3.04217072748092E-05
ENSG00000215472 -27.8483245149912 0.0397619153720593
ENSG00000215817 -13.5190841590305 0.00675659074205503
ENSG00000217236 -20.5956813104989 0.0287502245020191
ENSG00000221988 -15.437030075188 0.000881217599989722
ENSG00000221988 -12.5576953684546 0.0151972472985044
ENSG00000223547 -41.025641025641 0.048531701906358
ENSG00000223547 -41.025641025641 0.048531701906358
ENSG00000223547 -36.1852433281005 0.00120676448013167
ENSG00000224041 -26.5662172878668 0.0141737948892525
ENSG00000224470 -35.0862068965517 0.00826722019021418
ENSG00000232237 -18.4426964277711 0.00706947340347165
ENSG00000232434 -15.4621775059731 6.92750811815583E-05
ENSG00000233041 -25 0.0498242721317406
ENSG00000233436 -10.5533596837945 0.00500520808218314
ENSG00000236398 -23.7884872824632 0.0339178520847889
ENSG00000240505 -16.1968400674452 0.043591041794672
ENSG00000241697 -20.2541827541828 0.0197473266150249
ENSG00000243646 -15.8593909666871 0.0498537960889873
ENSG00000243943 -13.5289634146341 0.00548177529708554
ENSG00000244187 -12.7288319237544 0.000563775169064139
ENSG00000244187 -20.4971229760471 0.00570246594908727
ENSG00000254995 -12.3358585858586 0.00423793375162113
ENSG00000254997 -17.9655870445344 0.0315032058089109
ENSG00000255221 -25.547055684042 0.036555069792132
ENSG00000255690 -11.6808149405772 0.0189770213776895
ENSG00000255690 -12.3504896041879 0.000512147430381347
ENSG00000256463 -16.3964019723133 0.0248820738071412
ENSG00000256463 -21.898750249406 1.95468984574037E-06
ENSG00000256463 -24.0959541711421 0.000163854290307613
ENSG00000257520 -13.5327327327327 0.0236714264555738
ENSG00000258388 -15.437030075188 0.000881217599989722
ENSG00000258388 -12.5576953684546 0.0151972472985044
ENSG00000258644 -25.126213592233 0.0333275081725526
ENSG00000260596 -32.0075757575758 0.000264090097761395
ENSG00000262481 -25.2622377622378 0.0374908539061022
ENSG00000265681 -27.8483245149912 0.0397619153720593
ENSG00000267001 -19.2689677629437 0.0139942362377622
ENSG00000267110 -21.4364035087719 0.000722902618995026
ENSG00000267335 -24.5206349206349 0.00395336958051468
ENSG00000267477 -42.1052631578947 0.00358926842407119
ENSG00000267552 -18.7379567745855 0.00601386696065428
ENSG00000267631 -24.5206349206349 0.00395336958051468
ENSG00000267748 -11.7440499414748 0.0351157095258298
ENSG00000268163 -17.6767676767677 0.0134216004209188
ENSG00000268163 -20.2912102591504 0.0247558735443318
ENSG00000270800 -10.6719405397908 0.04406634342951
ENSG00000272896 -12.7288319237544 0.000563775169064139
ENSG00000272896 -20.4971229760471 0.00570246594908727
ENSG00000273049 -11.8653524270039 0.0285551186534443
ENSG00000273154 -13.2554549434345 0.0464070147864675
ENSG00000273259 -16.4386737769534 0.0364912699901172
ENSG00000273706 -13.5100521545781 1.51867451212391E-07
ENSG00000273749 -12.1543294020032 0.000191335185544997
ENSG00000277462 -14.5836288835296 0.00349485787643516
ENSG00000277462 -13.4898365882822 0.0171288842575121
ENSG00000278685 -16.6554459502971 0.0446906038244175
ENSG00000278685 -23.0212765957447 0.0282787667085562
ENSG00000282881 -23.4083850931677 0.0226508605392879
ENSG00000283189 -10.7424071991001 0.0174515769503486
ENSG00000283526 -18.0124833163225 3.86317235533345E-06
ENSG00000283526 -19.2275318817793 4.43243451267144E-09
ENSG00000283580 -15.6722709914199 0.025593910912043
ENSG00000283586 -10.150711301 0.0134787535587177
ENSG00000283586 -12.0691131760373 0.0371369270217128
ENSG00000284194 -12.589080993989 0.0188369897278606
ENSG00000284713 -11.5865202574782 0.0125498653646383
ENSG00000284713 -12.4206553439698 0.025610830019555
ENSG00000284906 -15.7713467817686 0.045902210404969
ENSG00000285077 -15.7713467817686 0.045902210404969
ENSG00000285471 -13.0089802670448 0.00513538343420379
ENSG00000285471 -15.86969610546 0.000385506270676657
ENSG00000288611 -10.381169344394 0.00449479933783133

Table 4.

Biological function of genes with elevated methylation levels.

GO:0030308/negative regulation of cell growth
ENSG00000100739
ENSG00000166923
GO:0008285/negative regulation of cell proliferation
ENSG00000088992
ENSG00000103197
ENSG00000135956
ENSG00000140105
ENSG00000165238
ENSG00000178568
ENSG00000182636
ENSG00000183145
ENSG00000198625
GO:0007050//cell cycle arrest
ENSG00000123999
ENSG00000162670
GO:0043065/positive regulation of apoptotic process;
ENSG00000064300
ENSG00000104765
ENSG00000109906
ENSG00000120063
ENSG00000131023
ENSG00000138613
ENSG00000145779
ENSG00000171680
ENSG00000176884
ENSG00000197442
Biological function of genes with decreased methylation levels
GO:0010720/positive regulation of cell development
ENSG00000005073
ENSG00000128422
GO:0008284/positive regulation of cell proliferation
ENSG00000037280
ENSG00000105550
ENSG00000105989
ENSG00000110148
ENSG00000119630
ENSG00000135452
ENSG00000135625
ENSG00000136630
ENSG00000169057
ENSG00000169083
ENSG00000170961
ENSG00000172201
ENSG00000180818
ENSG00000181104
ENSG00000184486
ENSG00000187094
GO:0043066/negative regulation of apoptotic process
ENSG00000071054
ENSG00000101188
ENSG00000106144
ENSG00000108839
ENSG00000134259
ENSG00000143479
ENSG00000143867
ENSG00000169230
ENSG00000172175
ENSG00000173530
ENSG00000188389
ENSG00000197785
ENSG00000198171
GO:0050728//negative regulation of inflammatory response
GO:0006979//response to oxidative stress
ENSG00000069667
ENSG00000128016
ENSG00000100983
ENSG00000122691
ENSG00000174130
ENSG00000184470

2.4. MSCs regulate gene transcription by inducing DNA methylation modification

To study the relationship between the DNA methylation modification pattern and gene transcription during MSCs reversal of TEC senescence, the genes in TECs were divided into four groups according to transcription level (high, medium, low and negative expression). We found that there was a negative correlation between DNA methylation and transcription in the promoter region (upstream 1 kb), a positive correlation between DNA methylation and transcription in the gene body region (Fig. 4A). In addition, the Pearson correlation coefficients (r) between DNA methylation levels and corresponding gene expression levels in the coculture group were calculated. The results also showed that DNA methylation levels in promoter and gene body regions were negatively correlated and positively correlated, respectively, with DNA methylation levels (Fig. 4B).

Fig. 4.

Fig. 4

MSCs regulate gene transcription by inducing DNA methylation modification. A In the process of reversing the aging of TECs via MSCs, the methylation level of different positions of the gene is associated with the transcriptional level (the abscissa shows different gene locations divided into upstream 1k, gene body and downstream 1k; the ordinate is the methylation level; the red curve indicates high transcription, the green represents low transcription, the blue curve represents medium transcription, and the purple curve represents no transcription). B Promoter (above) and gene body region (below) DNA methylation levels and corresponding gene transcription scatter plots (the red and blue curves represent gene transcription levels and DNA methylation levels in promoter and genomic regions, respectively). The horizontal axis below each box from left to right represents the level of gene transcription from high to low. C Nine-quadrant diagram of the relationship between DNA methylation and gene RNA-seq data in the promoter region (the abscissa is the RNA-seq log2fc value, the ordinate is the differential methylation level, and the upper left and lower right represent a significant negative correlation between promoter methylation and the transcriptional level). D GO enrichment analysis showed that the level of DNA methylation in the promoter region was negatively correlated with gene expression (ordinate: GO term, abscissa: number of genes; red indicates upregulated genes, and green indicates downregulated genes). E KEGG enrichment analysis of genes with decreased methylation levels. F The expression of RAPA1, SRC and ITGB3, the key proteins of the Rap1 signaling pathway (the measured values represent the mean ± standard deviation (`X ± s) of three replicate experiments. ∗∗P < 0.01, ∗∗∗P < 0.001). G A nine-quadrant map of the relationship between DNA methylation and gene RNA-seq in the gene body region (the abscissa is the RNA-seq log2fc, and the ordinate is the differential methylation level). H In the gene body region, the DNA methylation level was negatively correlated with gene expression and positively correlated with enriched GO terms (BNG indicates that the methylation level was negatively correlated with gene expression; BPG indicates that the methylation level was positively correlated with gene expression).

In the promoter region, after MSC coculture, 66 genes in senescent TECs exhibited a significant negative correlation between promoter methylation and transcription (Fig. 4C and Table 5). Methylation of PDE5A, DUOX2, LAMP1 and SVIL in the promoter region led to the downregulation of transcription. The expression of these genes mainly inhibited cell development, differentiation and the cell cycle. The demethylation of POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 in the promoter region led to an increase in transcriptional levels, and their expression mainly promoted the cell cycle, cell growth and development and epithelial cell differentiation (Fig. 4D). KEGG analysis showed that the genes with decreased methylation levels were mainly enriched in the Rap1 signaling pathway (Fig. 4E). Furthermore, the expression of key proteins RAP1, SRC and ITGB3 in the RAP1 signaling pathway was detected by Western blotting. The results showed that the expression of the key proteins RAP1, SRC and ITGB3 increased significantly after MSCs coculture, suggesting that the decreased methylation of PGF and NGF activated the RAP1 signaling pathway (Fig. 4F). The results showed that MSCs induced a decrease in the methylation level in the promoter region of ageing-promoting genes, promoted the demethylation of cell growth gene promoters, inhibited the expression of ageing-promoting genes, promoted an increase in cell growth gene expression, and finally reversed the senescence of TECs. The increased expression level of genes related to the RAP1 signaling pathway played an important role in this process.

Table 5.

Promoter methylation is negatively correlated with gene expression.

id meth.diff meth.q gene_fc gene_p
ENSG00000169393 12 0.0493167198534707 -3.16992500144231 0.0182157396451062
ENSG00000140279 11.3766298330057 0.0425112978457713 -3 0.0192457689020148
ENSG00000183496 14.5393689412893 0.000320527649816425 -1.77258950389693 0.00215297544876282
ENSG00000166147 15.6619833863194 0.0468581371125151 -1.07480383893417 5.03157126804725E-30
ENSG00000138735 16.2222222222222 0.0350319355454426 -1.03585333801322 7.42879931992132E-58
ENSG00000178568 27.5180140945443 1.07565396326703E-07 -0.888968687611259 0.0032937298473144
ENSG00000221890 22.6050420168067 0.0345626215065825 -0.690795412733281 0.0176007089136136
ENSG00000143412 12.5132555673383 0.0244041195439192 -0.651006139135347 3.21495919043423E-06
ENSG00000145536 15.5727366159021 0.0089577681604829 -0.64965904943743 2.45479952909829E-05
ENSG00000188153 12.1476617552851 0.0439126087216701 -0.616462780545589 5.88521426071701E-15
ENSG00000167861 25.9545923632611 0.00358556744024635 -0.600339911716719 1.51057836248098E-06
ENSG00000198691 16.1113699906803 0.0178381603850453 -0.587156714134073 0.000809594934608358
ENSG00000138336 21.8070362473348 0.000861502987558523 -0.587007426706185 0.000973023962220727
ENSG00000101751 15.6150627615063 0.00847306283066711 -0.556330347365361 0.00314889263219378
ENSG00000197321 26.6782608695652 0.0141433998222048 -0.501773106156611 5.87968999963141E-08
ENSG00000184731 13.2576254294631 0.0104233490346365 -0.431430376467744 9.26373589126763E-08
ENSG00000164920 13.8862559241706 0.00141583621004862 -0.390157290173661 4.66508235306963E-06
ENSG00000196498 23.0205156060669 5.350072520849E-05 -0.365364917610353 0.0024253152303974
ENSG00000110628 12.6218323586745 0.000905019819790039 -0.364197318260097 0.0233052964014367
ENSG00000111271 14.5110267534346 0.0359526622537845 -0.353001616041892 0.00560907003355677
ENSG00000149639 12.1855383969921 0.0160852095767229 -0.347636760659084 0.00218329338042741
ENSG00000170561 12.7706888335892 0.0391539116619081 -0.347476890469923 0.00333298319309075
ENSG00000224051 30.1339896089691 0.0291683709558524 -0.322945709676845 0.0220990443969277
ENSG00000175155 22.7486965589155 0.0111237785068831 -0.313192706891764 0.00257815783289071
ENSG00000185896 20.1874052638832 0.024131183824719 -0.270676478852593 1.05874659873463E-05
ENSG00000134259 -19.9652777777778 0.00459426665426082 1.83064557204383 0.00941141544514754
ENSG00000138944 -37.8117457763475 0.0169445660029793 1.37586690199816 0.00219311995651581
ENSG00000101216 -10.3382163724572 0.0370962647151181 1.35675568344045 1.7949955026023E-14
ENSG00000119630 -10.7784431137725 0.00695950565232784 0.888209174542443 0.00320217443092626
ENSG00000129654 -10.6407004024644 6.26010353840451E-06 0.795729596937978 0.0271087679454225
ENSG00000142273 -19.4395149364094 0.0341414149043605 0.74612559345168 0.0088904789182297
ENSG00000165799 -19.8527720266851 0.0447136844752293 0.678071905112639 0.0177003751106127
ENSG00000157193 -10.0792079207921 0.00307962939304754 0.666209101707102 5.04376339464156E-11
ENSG00000181104 -13.6891228909896 0.0435232730481889 0.621100205187945 3.5853266552734E-08
ENSG00000143479 -13.636249655207 0.0401229285764378 0.606004916283042 0.00637638237944121
ENSG00000128422 -13.7385720060132 0.0117581512531377 0.539551979134848 2.51234780697392E-20
ENSG00000005189 -14.1243862520458 0.0117387368539422 0.529489164822724 7.66287517170509E-05
ENSG00000163565 -11.0395892164983 0.00942619045704688 0.515773668044346 0.000690038898638332
ENSG00000037897 -13.2831444200079 0.0266330771143665 0.513225279513817 0.00221165164613025
ENSG00000188807 -14.1644210192597 0.00214158534118136 0.50354766909851 2.372630415506E-05
ENSG00000134686 -16.0714285714286 0.0353876886095324 0.49406131874452 5.10831089048732E-11
ENSG00000123130 -37.3015873015873 5.00924521270897E-06 0.489750774803252 0.00176615938851862
ENSG00000156398 -13.6112071947423 0.0442108996079291 0.487008729785849 0.0128476275418884
ENSG00000167470 -10.062893081761 0.0449721395775338 0.476336091099384 5.09652754620344E-09
ENSG00000173530 -16.4157644250816 0.0412017199604227 0.468398314663583 0.000143462486375593
ENSG00000167523 -16.751269035533 5.8315626359607E-08 0.464668267003444 0.0158976675818633
ENSG00000118985 -15.2974664454833 0.0372468840756965 0.409987808996061 4.97570168541322E-07
ENSG00000184445 -28.9085545722714 1.10468522189305E-19 0.397728662866405 9.03292391205679E-06
ENSG00000167513 -18.2418326468431 0.00513538343420379 0.395126956365581 3.04930833769411E-05
ENSG00000113356 -30.4485219164118 0.0128237638132768 0.390375445044573 0.0389512379357209
ENSG00000273706 -13.5100521545781 1.51867451212391E-07 0.389539574228011 0.0429548369026055
ENSG00000196923 -10.1846285959639 0.0125269750370792 0.375828051950098 0.00067391254848192
ENSG00000087269 -11.5588273279091 0.0292988774562968 0.359962553043222 0.00139454612866975
ENSG00000003400 -13.0225905823676 0.0261886189599571 0.357439579522302 0.00293504239997059
ENSG00000172175 -13.6658199347632 0.0450575089860559 0.341152662152261 0.0324220388410544
ENSG00000127586 -23.5960889504197 0.0106052756412346 0.339380789036883 0.0444323090993345
ENSG00000169230 -22.6702952333828 0.0422632036507693 0.314585152068477 6.08022403373139E-07
ENSG00000197451 -11.3869559539578 0.00256448161697349 0.309912043537486 6.84334406811732E-06
ENSG00000131620 -23.3758708893317 7.69815899489769E-05 0.303751434937819 0.00347391075687533
ENSG00000132773 -18.0035650623886 0.00122867731832998 0.287830317680935 0.0230792818269578
ENSG00000172009 -24.4159883528555 0.00479288133087388 0.287040237001174 0.00229691390720688
ENSG00000142731 -21.0726198609492 0.000484552422247172 0.282650503348821 0.00173861229602644
ENSG00000204599 -15.4369398207426 0.0188931632771714 0.27892876502747 0.0479497582826186
ENSG00000171824 -12.1212121212121 0.0220298071571259 0.276012259832004 0.00631234215092042
ENSG00000165688 -12.2080440996325 1.88001695122534E-05 0.27115480052629 0.000390441450012896
ENSG00000285077 -15.7713467817686 0.045902210404969 0.266423004081944 0.0468961829118312

In the gene body region, there was a significant negative correlation between methylation level and gene transcription in MSCs-cocultured senescent TECs, and these genes (534 risk-related genes) were defined as BNGs (Fig. 4G and Table 6). These genes are mainly involved in biological processes such as cell population proliferation, epithelial cell differentiation and regulation of epithelial cell proliferation (Fig. 4H). However, there was a significant positive correlation between the methylation level of more genes (553 risk-related genes) and gene transcription, and these genes were defined as BPGs (Fig. 4G). Interestingly, BPGs also had biological processes similar to those of BNGs (Fig. 4H). These results suggest that methylation changes in the gene body region may not only inhibit gene transcription but may also promote gene transcription in the process by which MSCs reverse TEC ageing.

Table 6.

genebody methylation is correlated with gene expression.

id meth.diff meth.q gene_fc gene_p
ENSG00000140379 17.1726137641281 0.0460288318272713 7.75933340719466 0.0367406623189661 BPG
ENSG00000198626 24.7863247863248 0.0428569231124602 5.4374053123073 0.0297721172788166 BPG
ENSG00000273217 12.0838262633091 0.0017609206903326 5.32192809488736 0.0445013525732622 BPG
ENSG00000185339 45.2941176470588 0.0247584438639287 4.3037807481771 0.00178271206325163 BPG
ENSG00000047457 13.4175878772823 0.0283611057261894 3.22994977251453 1.66788126916758E-24 BPG
ENSG00000288520 36.7167919799499 0.00179587138482442 2.7004397181411 0.0288626152504842 BPG
ENSG00000152953 35.8585858585859 0.0108455530672616 2.32192809488736 0.0352806015308715 BPG
ENSG00000197641 12.020202020202 0.0382074798208661 1.92619869958988 1.92719648884193E-25 BPG
ENSG00000198768 10.8635026873542 0.0455223482400552 1.91753783980803 0.0395432526802723 BPG
ENSG00000149557 30.7784252203795 0.0362978205542385 1.73142547723426 1.52589890721511E-08 BPG
ENSG00000081985 21.5675057208238 0.0323846219542733 1.72582503656101 0.0427904082297945 BPG
ENSG00000162711 17.6871052985175 0.000259725399986543 1.53605290024021 2.06547568623394E-07 BPG
ENSG00000259207 19.1260923845193 0.0176749247514635 1.49185309632967 5.16678595835762E-10 BPG
ENSG00000139278 10.7863631180875 0.000163880514573194 1.40892736291598 1.16755566875088E-14 BPG
ENSG00000154678 18.3439422018102 0.0436144473018669 1.39854937649027 0.0282987501155144 BPG
ENSG00000130164 21.9712015888779 0.0293316809944489 1.38476472870143 1.68437434910057E-68 BPG
ENSG00000288602 10.0578466131741 8.69490707846433E-06 1.38133628405012 1.22193778792156E-06 BPG
ENSG00000101216 14.0663390663391 0.00599990842666912 1.35675568344045 1.7949955026023E-14 BPG
ENSG00000163485 22.2225941422594 0.000337583593079858 1.34513548604869 0.0424124417976442 BPG
ENSG00000188001 19.9379675241744 0.0462884117852547 1.33763299559323 1.51293275212706E-05 BPG
ENSG00000090339 21.8632075471698 7.55280953546309E-10 1.22517486200778 1.17341214937116E-27 BPG
ENSG00000196639 10.9243697478992 0.0226627811165279 1.12687176584051 2.79229839886979E-11 BPG
ENSG00000002587 12.3692427790788 0.0030212729838875 1.09953567355091 0.00241065519046194 BPG
ENSG00000167914 24.1176470588235 0.00878621065548137 1.0790715709912 0.00212095639712 BPG
ENSG00000255398 10.5249083129584 0.00555500233808119 1.0759488532333 0.0213799392158058 BPG
ENSG00000124496 11.8697478991597 0.0197051200937206 1.05475976867859 9.74227997345186E-09 BPG
ENSG00000185022 21.4285714285714 0.00027811976367497 1.03023961317768 4.14560461289623E-22 BPG
ENSG00000112096 14.5454545454546 0.046217877185254 1.00829655641631 0.0277179645543597 BPG
ENSG00000138316 17.4206525733516 0.0304301935480177 1 0.00942259240403998 BPG
ENSG00000118515 11.5896851801896 0.000186787297261282 0.986540356802792 1.56070357468565E-40 BPG
ENSG00000125730 22.6524064171123 0.0001539492364998 0.940197017497937 1.10578488599274E-13 BPG
ENSG00000164120 28.8961038961039 0.0361783351821684 0.934782340553516 0.0395141713878454 BPG
ENSG00000163568 11.2735708508918 0.0279618332417633 0.931046368249858 7.94510291344592E-10 BPG
ENSG00000182752 27.8974358974359 0.0415386692927686 0.883355081147731 1.45670435422431E-15 BPG
ENSG00000111700 10.3794923347575 0.0474623507181872 0.867939147909315 0.0025682878525908 BPG
ENSG00000100379 10.7658959537572 0.015640943861462 0.8600179616186 3.78521809513934E-05 BPG
ENSG00000198121 11.5673799884326 0.000235463010867662 0.853133922996195 2.54618474694495E-12 BPG
ENSG00000129521 25.5984555984556 0.0222067965182979 0.84358980503217 2.59806804251617E-05 BPG
ENSG00000100060 21.0711768851304 0.0106912163846665 0.828631581688019 0.0273481690302178 BPG
ENSG00000075131 19.4487577639752 0.0424731991250545 0.816530118457942 0.00474497942871165 BPG
ENSG00000114541 28.8600288600289 0.0474421974610935 0.815747086313056 1.42922170794235E-22 BPG
ENSG00000174640 16.9152167909417 0.0387405060017022 0.800012946716431 0.00209466071836222 BPG
ENSG00000129654 16.5985021511659 0.0298086831017226 0.795729596937978 0.0271087679454225 BPG
ENSG00000156299 22.9379548628979 1.07161511929475E-05 0.792603966282472 5.65041318074885E-06 BPG
ENSG00000105137 10.7254534083802 0.04654172520713 0.786429083203057 0.000279118337134963 BPG
ENSG00000095303 16.1793847253354 0.0211069184576635 0.764794459829562 1.5199467943708E-06 BPG
ENSG00000134954 23.1418370658877 0.0277963868963604 0.761605766840066 2.56715039043354E-44 BPG
ENSG00000103546 16.2875313398153 0.00349398264893309 0.758806642705791 0.0418443211370969 BPG
ENSG00000160256 13.6070021316453 0.0214647227421257 0.753891509689743 0.0332320116864089 BPG
ENSG00000164023 21.25 0.011922179126476 0.746225542778568 0.000363807452821192 BPG
ENSG00000123358 15.8217347220942 2.16626464371985E-05 0.737561626003184 5.9249188956385E-06 BPG
ENSG00000157654 15.244622339966 0.0376927615809148 0.735084635567444 0.00032002751951902 BPG
ENSG00000104856 25.2747252747253 0.000251979891967708 0.724771694854199 1.12785792226896E-07 BPG
ENSG00000138829 38.4102564102564 0.0495299856395462 0.713477241243836 0.0417689085213096 BPG
ENSG00000198517 10.0071965456581 0.000250857317643525 0.710892082556315 1.95937520854343E-12 BPG
ENSG00000140525 12.7136879856545 0.0492349249753731 0.703928500914718 1.88197511468814E-10 BPG
ENSG00000034152 26.2759170653907 0.00144846259665856 0.693086631049928 5.24816371136761E-05 BPG
ENSG00000213397 26.1435608726249 0.0272650692982965 0.689932060322699 0.000404367736333311 BPG
ENSG00000184408 26.011760636458 0.039074500980129 0.667424660913128 0.0384781428522723 BPG
ENSG00000077238 12.7857142857143 0.00257455533047546 0.662159614919003 1.55807095924066E-27 BPG
ENSG00000116396 26.0964408725603 0.0096893888071383 0.657112286476989 1.35258473501439E-06 BPG
ENSG00000145220 23.3350488934637 0.00284511941794192 0.652682641836568 0.000888214720779499 BPG
ENSG00000135074 28.6655462184874 0.00818970336907842 0.650885862801071 1.17888754456392E-37 BPG
ENSG00000008517 14.6666666666667 0.0123730750888345 0.648782870854652 0.0124472970165069 BPG
ENSG00000134070 16.1204481792717 0.0327234089529168 0.642404392962848 0.00154731554224759 BPG
ENSG00000130066 19.1561875661065 0.0406547451253858 0.639525806945979 0.000483308213269925 BPG
ENSG00000115109 17.8514588859416 4.29516076726877E-06 0.631074425575469 0.0210371778011025 BPG
ENSG00000006432 28.8508118776352 0.00145775249728922 0.629872816855305 2.88466436771349E-27 BPG
ENSG00000126091 12.0560248048754 0.0298997792651608 0.626850897011178 0.00839083915114877 BPG
ENSG00000185033 12.3076923076923 0.0246845329093198 0.625898044084284 2.59811443497528E-21 BPG
ENSG00000073756 11.7949854686961 4.76806497395308E-10 0.624200327276437 4.92058830388309E-17 BPG
ENSG00000127666 20.7014835777378 0.0361625798985242 0.617666665793861 2.82406615980706E-06 BPG
ENSG00000171612 10.2189781021898 0.0299114399803943 0.616403847923919 3.85613698182859E-06 BPG
ENSG00000188352 14.4671841919548 0.0234512792736483 0.616017225301995 1.77958293421601E-09 BPG
ENSG00000154217 10.3516533749092 0.000322810648896246 0.615899482470343 0.000662697989781469 BPG
ENSG00000112159 14.5891528611334 0.015561117088738 0.610708775591004 0.00592967658047154 BPG
ENSG00000143479 19.2099637918248 0.0216185651681438 0.606004916283042 0.00637638237944121 BPG
ENSG00000250021 11.3825232938806 0.0240344121618717 0.593732710348888 0.00848014211495732 BPG
ENSG00000128294 10.5763517528223 0.00140043111047096 0.59321592735501 0.0345827448979271 BPG
ENSG00000154655 18.9600840336134 0.0123963264712088 0.59183345241 1.03930323127386E-06 BPG
ENSG00000165650 18.3908045977011 0.0015304926249123 0.579453598291972 0.0453755163033109 BPG
ENSG00000179583 23.2768127929418 0.0270751116053932 0.577751585826059 0.000353913888725189 BPG
ENSG00000140945 37.7016129032258 0.00981463319013045 0.574978412148533 0.00366332390862918 BPG
ENSG00000128578 10.0450605262153 0.0130192453752586 0.574341384964587 0.00284139827758737 BPG
ENSG00000131941 19.8850447662209 0.00447847862477616 0.573258396588686 1.74407845148977E-14 BPG
ENSG00000175832 11.9083269671505 0.0394262915203526 0.571278916880122 0.000200810502007593 BPG
ENSG00000166444 17.8225806451613 0.0194673305790636 0.56545984417481 4.99627819602438E-09 BPG
ENSG00000156113 26.7635590216235 0.0105659788754113 0.562791119722535 0.0227562050853387 BPG
ENSG00000142185 51.7641129032258 8.09380543598331E-08 0.561438218660123 0.0293332076847108 BPG
ENSG00000145675 22.5126646403242 0.0137608208710987 0.55549389265715 0.02168041321141 BPG
ENSG00000133773 23.4776987718164 0.0497599820915567 0.553529152545131 0.0232769540006936 BPG
ENSG00000115641 17.0194508009153 0.0110685012966746 0.552391837873705 1.0526762753976E-29 BPG
ENSG00000060558 13.75 0.0266218044809461 0.550616755318472 0.00025669154956774 BPG
ENSG00000147065 29.0869916509561 0.0240709907229761 0.53654301642417 8.11287354734939E-30 BPG
ENSG00000110427 36.6200578592093 0.0122666642572542 0.532060599558595 0.00291374882581983 BPG
ENSG00000159200 13.9405684754522 0.0441669320419815 0.529082695095812 7.08904757466765E-07 BPG
ENSG00000116120 13.2267732267732 0.0344536764202843 0.528875755953478 0.00805916733707207 BPG
ENSG00000162419 27.9619805481874 0.00685307479690918 0.525155335489124 0.00223599875365847 BPG
ENSG00000167670 21.763859832636 2.73646856659351E-07 0.521039303621415 0.000147383634918683 BPG
ENSG00000005884 22.8070175438597 0.0402615281928553 0.513443633410088 3.7351721134188E-23 BPG
ENSG00000171488 17.1157687795288 3.2058714522525E-07 0.513239813177653 0.00114332064975316 BPG
ENSG00000073712 12.5 0.0341240726600526 0.512037234330269 1.28218578484813E-07 BPG
ENSG00000137309 15.2883630986236 0.00729589058035453 0.511487559636413 4.36463899514817E-15 BPG
ENSG00000125731 17.948717948718 0.0396708027087845 0.509667175183969 0.00391499527626842 BPG
ENSG00000196792 12.5 0.0439863781029824 0.507954281998884 0.0461085059867358 BPG
ENSG00000101773 27.5862068965517 0.0387651384224958 0.506702061037264 1.17953929546148E-06 BPG
ENSG00000134686 11.9293924466338 0.0319547070303023 0.49406131874452 5.10831089048732E-11 BPG
ENSG00000125454 16.9843160611475 0.0163651212626911 0.494043665158886 0.00543931724489655 BPG
ENSG00000285901 11.6166587674845 0.0444463457358793 0.485665901665924 0.00265331917593365 BPG
ENSG00000075702 23.7220969376083 3.0737873482305E-10 0.484470132810983 0.0150733519063799 BPG
ENSG00000039560 13.9867156739778 0.00138145335108338 0.483171930628531 6.66514055683011E-11 BPG
ENSG00000168970 11.4285714285714 0.0459755722858929 0.48275764039426 0.0452815641249222 BPG
ENSG00000100023 12.4508215690812 0.0133970134671646 0.482668327207678 0.0105207229595894 BPG
ENSG00000132334 12.1472244811903 0.0474686841805455 0.475178385333266 7.6377217442228E-05 BPG
ENSG00000073282 11.9007689143436 0.048546782398754 0.473557736578936 0.00196863331449595 BPG
ENSG00000177706 17.7135047693423 0.00787495879534455 0.471523213919535 5.28191185180264E-07 BPG
ENSG00000100065 26.4326432643264 0.0191484913378948 0.470268230654617 3.15996374866782E-08 BPG
ENSG00000198042 17.7547169811321 0.0232281921397903 0.468902336950411 8.33417033982992E-09 BPG
ENSG00000173530 10.1123595505618 0.0384166465261006 0.468398314663583 0.000143462486375593 BPG
ENSG00000137478 15.511611030479 1.51079661917468E-05 0.46837326134002 0.00725447648208387 BPG
ENSG00000171132 24.9302501536861 0.0160456878041116 0.468248157279246 0.0072417886941382 BPG
ENSG00000149177 10.7246688919996 0.00630639306502788 0.467617414400212 0.0446129236346782 BPG
ENSG00000113643 10.2938593409682 0.00576526528660252 0.46607475129958 0.0101843122334553 BPG
ENSG00000109320 15.1220905326289 0.00163633849319427 0.460363788572407 6.35552267042898E-06 BPG
ENSG00000134775 12.1574777154176 0.00829919587995446 0.459090918425327 3.0528769923768E-05 BPG
ENSG00000169851 21.8632075471698 0.0431605350915495 0.453708983530555 5.70757756837102E-06 BPG
ENSG00000259316 23.1493553527452 0.00128148450740161 0.451443145707578 0.0297853829323746 BPG
ENSG00000128050 14.9583297534152 0.0158462688766661 0.451141139038848 5.93185210126709E-16 BPG
ENSG00000179958 14.7819042555885 2.14522521012426E-05 0.44825768623875 3.09507657388312E-08 BPG
ENSG00000157551 11.5706200154361 0.0419093860693028 0.447594054452798 0.00230304213579688 BPG
ENSG00000163866 23.685580426861 0.049655105392165 0.44323975577384 0.000258724968656793 BPG
ENSG00000196793 11.978766501362 0.0460453262119517 0.435328215579876 0.00221912060149269 BPG
ENSG00000135763 16.8818129661503 0.0302759054994361 0.433984742554527 6.53126311486347E-08 BPG
ENSG00000092969 11.648993391732 0.0210412227946416 0.432025567700634 1.3965214654066E-08 BPG
ENSG00000081181 15.3088177871695 0.0430246629965016 0.431315577801243 0.0498940329939293 BPG
ENSG00000148459 14.4086021505376 0.048742111856042 0.429364974832301 0.0433078449692255 BPG
ENSG00000051180 16.4885639295088 0.0420749503776668 0.427558428562299 3.10366858073935E-05 BPG
ENSG00000113119 14.2857142857143 0.0395734332048679 0.424497828527911 0.02981061954876 BPG
ENSG00000100311 27.8616120721384 3.50691882283434E-07 0.424012807497665 8.81366769431761E-06 BPG
ENSG00000126261 13.347296451442 0.000867938947185886 0.421711643478361 3.50063286508391E-07 BPG
ENSG00000155903 13.953488372093 0.0018964907491042 0.42148143218127 0.0102460084903322 BPG
ENSG00000120254 10.3525152439024 0.00242563790018186 0.416225112127262 3.11356337626985E-05 BPG
ENSG00000126215 18.6881263285758 0.0047041318130588 0.416095969642456 2.96320540656788E-06 BPG
ENSG00000159131 20 0.0337146988601268 0.414334455978619 1.40212757091001E-07 BPG
ENSG00000197903 11.3590263691684 1.80362232316675E-09 0.410538463717116 7.24747897837557E-06 BPG
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ENSG00000196935 -12.5095492742552 0.0312581984774442 -0.317212837258018 6.26141169093279E-06 BPG
ENSG00000105810 -17.3458725182863 0.0154453795157378 -0.316635644920549 0.0001567758234917 BPG
ENSG00000179912 -10.8843537414966 0.00435216859219888 -0.315746578471056 0.000874410459462611 BPG
ENSG00000112144 -12.3076923076923 0.0352358160993718 -0.313903923802541 0.00138892875889279 BPG
ENSG00000196628 -26.4150943396226 0.00159773648008419 -0.313420129875148 3.28353137619013E-05 BPG
ENSG00000175155 -10.767405236335 0.0492552564194126 -0.313192706891764 0.00257815783289071 BPG
ENSG00000131149 -72.3484848484848 6.02086208344641E-07 -0.312479031368485 8.88086989224702E-05 BPG
ENSG00000173402 -17.3076923076923 0.00259101568638736 -0.311495771507253 6.72980902479292E-07 BPG
ENSG00000172817 -13.0865201372126 0.0148212401623412 -0.310860790984529 0.000193135720485011 BPG
ENSG00000033122 -15.1976099351714 0.0443638347308002 -0.306925931548116 0.00649183678214571 BPG
ENSG00000188064 -12.2101449275362 0.0219575344210156 -0.30632285680212 0.00403868260498254 BPG
ENSG00000145431 -11.3122820427077 0.0245549610355889 -0.304810542546114 0.0331405587117779 BPG
ENSG00000168795 -10.063648762746 0.035655980992928 -0.304780788272944 0.00124804558314024 BPG
ENSG00000152104 -19.5183486238532 0.0186903174950322 -0.304201627678951 9.3801020593915E-09 BPG
ENSG00000171517 -10.7142857142857 0.030254997250585 -0.303203453090096 0.027909916119053 BPG
ENSG00000148498 -25.6010171058715 0.00121364071948905 -0.302775645054516 0.000599403897821241 BPG
ENSG00000109654 -37.5 0.0341617113401962 -0.301685799799022 0.0170642350636622 BPG
ENSG00000107104 -10.3243861338469 0.0431702429948203 -0.300742764996734 0.00633350140038271 BPG
ENSG00000130844 -39.983164983165 4.16525166325114E-10 -0.300668723482284 0.00719998180056289 BPG
ENSG00000217128 -11.6228070175439 0.0495363996427847 -0.298728320110066 9.04052804294962E-05 BPG
ENSG00000091157 -13.7931034482759 0.0109254916292158 -0.297059220774465 0.0374840228006454 BPG
ENSG00000144306 -17.2854441575341 0.00559347748568485 -0.293767533373402 0.0228695802793786 BPG
ENSG00000146648 -15.6407144095717 0.037114166441291 -0.293703786413115 2.29989877751664E-08 BPG
ENSG00000013288 -20.1121794871795 0.00270720181302578 -0.293473449710851 0.000421015976627107 BPG
ENSG00000075568 -21.9716494845361 0.0419162189190783 -0.293035982058586 0.0302709234446858 BPG
ENSG00000163701 -11.1111111111111 0.0356666493579973 -0.292553492753849 0.0388021610140557 BPG
ENSG00000105426 -19.0459067481721 0.0115227968479095 -0.29136312889229 9.61011029442332E-06 BPG
ENSG00000142784 -15.6462585034014 0.0170921943617 -0.291309926911496 0.000705471601159669 BPG
ENSG00000213694 -15.9353471118177 0.000600072464513817 -0.289866885655064 0.00708301690362163 BPG
ENSG00000119711 -20.3245942571785 0.00640464663479342 -0.289825497682747 0.0011433293486166 BPG
ENSG00000124587 -10.219068956933 0.0480419481129848 -0.289260874309599 0.0126463946039842 BPG
ENSG00000122779 -23.0899830220713 0.0338399491511729 -0.286905809452637 0.0312074493440943 BPG
ENSG00000133424 -29.8483736854977 0.00295502009273725 -0.285218427692496 0.0188359404674102 BPG
ENSG00000164073 -15.3201571890892 0.0150902906467985 -0.283527577697872 0.0348579282153239 BPG
ENSG00000196743 -15.7559198542805 0.00253350944854052 -0.28258280221177 0.00252542188050536 BPG
ENSG00000133983 -18.4473929236499 0.000409714520048529 -0.28200331050987 0.0264347269628561 BPG
ENSG00000137802 -29.5496991076987 0.045919863933346 -0.279574081983723 6.17422330703431E-07 BPG
ENSG00000177426 -16.8514136992784 0.0019906890357851 -0.279003057669837 4.43854792534203E-05 BPG
ENSG00000139266 -26.486915146709 2.18453721103963E-13 -0.277918279325949 0.0404238161915395 BPG
ENSG00000183018 -15.8710730314158 0.0303798945817675 -0.275184263619617 0.0380516157947918 BPG
ENSG00000125637 -19.8146513680494 0.0255341249935498 -0.272566168006445 0.0497067351784928 BPG
ENSG00000197757 -11.0945824396319 0.0237079682771938 -0.271302021817394 0.0131892309227297 BPG
ENSG00000177853 -11.0696612105063 0.00840131928524951 -0.268605858957599 0.0290459942156785 BPG
ENSG00000054965 -15 0.0466784626955542 -0.267963637356679 0.000293100155619997 BPG
ENSG00000148842 -11.4588050599281 0.0266352962539337 -0.267695783203657 0.00377595052802898 BPG
ENSG00000123636 -28.1315458734814 0.0140819601108139 -0.267416852462943 0.011351823485927 BPG
ENSG00000126858 -12.2970639032815 0.000516604023102541 -0.266499681624815 0.0311298311886331 BPG
ENSG00000162434 -20 0.033302838632579 -0.263612329451374 4.94379178207903E-08 BPG
ENSG00000243708 11.4285714285714 0.0459755722858929 -10.2086408085582 0.0303914223244126 BNG
ENSG00000285791 11.6889326831308 2.58663260106533E-05 -5.32192809488736 0.0444464233284871 BNG
ENSG00000284686 15.2453424298085 0.0142705765108504 -4.56478461878353 0.0159534725530422 BNG
ENSG00000284554 20.4896907216495 0.0239166953197774 -2.88452278258007 0.0161004908719089 BNG
ENSG00000141052 23.4808612440191 0.0332321208951147 -2.84130225398094 0.00445823481164802 BNG
ENSG00000198673 34.2760180995475 0.0471210718080951 -2.67807190511264 0.0262071277264242 BNG
ENSG00000166509 23.8829833203699 0.000107100204342874 -2.44166733916145 9.2409671837753E-10 BNG
ENSG00000172346 13.8792102206736 0.0338485749067207 -2.30666133823405 0.00785735661929826 BNG
ENSG00000055163 18.4180790960452 0.0478510307688422 -2.2829339632715 0.0165454370593668 BNG
ENSG00000144218 26.7397628104721 0.0231025020147638 -2.17791779219585 1.12979214761745E-05 BNG
ENSG00000085741 21.8053804119378 0.0099531998749705 -1.96578428466209 0.000496517358048221 BNG
ENSG00000122367 14.6890216261722 0.000179835622794449 -1.85561009066482 0.00523883401102577 BNG
ENSG00000159450 11.9856933865753 0.0244013429886495 -1.85414913353655 0.00369912599429018 BNG
ENSG00000170871 20.7547169811321 0.0356584465615654 -1.66699826958717 4.56446949371295E-10 BNG
ENSG00000145526 27.8409090909091 0.0265871205051091 -1.61598939634178 0.00646044956681184 BNG
ENSG00000184588 21.6815809097688 0.0320487792308055 -1.6000299488101 3.46814360898919E-07 BNG
ENSG00000177679 27.6068965517241 0.0141703113104466 -1.59991284218712 0.0138522073197324 BNG
ENSG00000115844 19.5057536313903 0.023166417767195 -1.4723706743448 0.00193013004584154 BNG
ENSG00000134516 22.2453646981949 0.0240135392868345 -1.41503749927884 0.018051969347599 BNG
ENSG00000137834 11.8944143630664 0.0161432184490245 -1.4039992278264 1.34050871745801E-05 BNG
ENSG00000164292 19.9233716475096 0.0364557999486495 -1.32775695358668 2.06829375217998E-43 BNG
ENSG00000110693 20.5844828001252 0.0214335484821773 -1.3112016882943 0.00661793779747204 BNG
ENSG00000031081 11.8811881188119 0.0285139918378 -1.30618535834161 0.0136161269955364 BNG
ENSG00000104081 14.6915584415584 0.0266766311662972 -1.28504761764604 2.89295613727264E-29 BNG
ENSG00000179869 14.2641077523658 0.036012662655069 -1.27077305420077 2.96061122587694E-26 BNG
ENSG00000183307 32.9376854599407 7.37254582691034E-06 -1.25779775746765 0.0201283284961805 BNG
ENSG00000125378 23.908804559772 0.0141634247950583 -1.22239242133645 0.00421868324091519 BNG
ENSG00000187720 13.1598457888494 8.52728379619754E-05 -1.13814629276965 0.012520318473065 BNG
ENSG00000143341 17.9016221873365 0.0235758336459982 -1.13670923392942 6.97523100767761E-36 BNG
ENSG00000112902 27.8081467113384 0.0190729888469772 -1.11393198503502 1.05408010797426E-37 BNG
ENSG00000050628 29.1620207564971 0.0142390504680928 -1.11103131238874 0.0159352652065548 BNG
ENSG00000164125 11.1111111111111 0.0114068205386867 -1.0994888016056 2.13172674056113E-29 BNG
ENSG00000172348 35.0877192982456 0.0256081938719352 -1.09019780897158 0.0423127348056044 BNG
ENSG00000166147 36.1985472154964 0.0302694806912422 -1.07480383893417 5.03157126804725E-30 BNG
ENSG00000168477 24.7942386831276 0.000309416960514426 -1.07297944535711 4.18654241470581E-09 BNG
ENSG00000227051 19.4767441860465 0.00156131261208117 -1.06558834162758 0.0116318181984038 BNG
ENSG00000204947 10.9382151029748 0.0400885775398988 -1.05794734920928 0.00408853581326887 BNG
ENSG00000178026 29.3280632411067 0.0436981213068628 -1.03963178689293 0.0125500731349175 BNG
ENSG00000130701 12.666383460776 0.0374981214266512 -1.03652587602511 0.00215566552074285 BNG
ENSG00000069702 13.8827838827839 0.0270751116053932 -1.02537178970831 1.85966982990304E-10 BNG
ENSG00000168916 13.0047371982856 0.00666888599889314 -1.02291693739155 0.0298227184961054 BNG
ENSG00000116039 21.7977019301523 0.0182959230355484 -1.01495034146597 0.00147837289196474 BNG
ENSG00000157502 10.7212071629852 0.0298875776396145 -0.99885817561045 1.63962018298735E-17 BNG
ENSG00000097096 11.0371346956713 0.0175081999482966 -0.996406735275991 1.53173247911141E-18 BNG
ENSG00000197183 15.8699862994284 0.0248089139195588 -0.987198717776908 0.0149582145196133 BNG
ENSG00000204103 12.66446797431 0.000520207556120523 -0.979535897440284 0.0445563180502833 BNG
ENSG00000182263 13.5664335664336 0.0044007150689271 -0.97480591784075 3.43643082167393E-14 BNG
ENSG00000111799 16.685280321644 0.0328568094477351 -0.951255666148583 1.60507703090466E-13 BNG
ENSG00000146477 18.4911717495987 0.0194503531162814 -0.936947829464703 0.000353422946928915 BNG
ENSG00000177076 15.863840719332 0.0144405638821234 -0.929610672108602 3.49843978402604E-12 BNG
ENSG00000126803 10.5054798516481 0.00219758317404919 -0.927718158319253 1.22276845523072E-24 BNG
ENSG00000114166 17.0500560006892 0.0188813032991278 -0.926874802389804 2.55900871212831E-07 BNG
ENSG00000167183 13.1452244989363 0.0112001369917942 -0.921163842447899 1.5003407140692E-08 BNG
ENSG00000185432 13.8783109080139 0.04153131178274 -0.918437760189343 1.3451990699202E-10 BNG
ENSG00000178568 37.0017128620367 2.62446043121715E-21 -0.888968687611259 0.0032937298473144 BNG
ENSG00000151623 11.9830450223393 0.0156272601196254 -0.888830624063527 2.85802238792563E-05 BNG
ENSG00000245848 28.8480263650663 0.000588322424264537 -0.882292014433902 1.19753165707245E-06 BNG
ENSG00000150893 12.7647612756328 0.0193133285667364 -0.867784804610925 2.03480136814508E-10 BNG
ENSG00000165029 15.7516412595972 0.0429688266413051 -0.86291825524471 0.000454633977955596 BNG
ENSG00000078018 18.6377708978328 0.0267348110796337 -0.856913044052906 0.00025794820431816 BNG
ENSG00000087085 20.9770114942529 0.0099341108704103 -0.841770332190061 0.000518237003680987 BNG
ENSG00000104205 10.0578466131741 8.69490707846433E-06 -0.837798814548365 1.07967548935029E-05 BNG
ENSG00000088002 41.5165165165165 0.0104801422938026 -0.82505692379106 0.0298353550538748 BNG
ENSG00000185924 11.3515115589804 0.0318484916420642 -0.797562185039193 0.000271873678644402 BNG
ENSG00000066468 31.0344827586207 0.00473328658620488 -0.79071950242535 7.93144139610672E-21 BNG
ENSG00000185352 15.4640921409214 0.0361105332861815 -0.79007693062577 0.0447431224531728 BNG
ENSG00000125848 14.0999692559951 0.0314930098693842 -0.786090105274825 0.0237497881247026 BNG
ENSG00000168453 16.1601597160603 0.0167552169351298 -0.752291325395042 0.00224328328413343 BNG
ENSG00000011201 34.5980126467931 0.040528242942424 -0.750374039136391 5.83095093384214E-05 BNG
ENSG00000176182 16.4007826021522 0.00102380692089381 -0.741364058063724 0.0382622065004552 BNG
ENSG00000139998 13.5398054397459 0.0302652271862405 -0.73884967913656 3.44175359709653E-16 BNG
ENSG00000253910 16.2661776264082 0.00844151005684433 -0.72950014391964 0.0396713817830189 BNG
ENSG00000196872 12.6008851861494 0.0459907985460533 -0.727960780904998 1.35353238986487E-09 BNG
ENSG00000126217 18.4417245158933 0.0478245948927844 -0.721570218766501 5.08518675875215E-07 BNG
ENSG00000165861 17.776590937102 2.8745872305254E-07 -0.720961014729347 7.00257301272694E-09 BNG
ENSG00000256223 18.1818181818182 0.0247394474772389 -0.715820262853991 0.000393372988416042 BNG
ENSG00000120093 14.7095375722543 0.0307865686470777 -0.714467624516055 0.0242160980884065 BNG
ENSG00000108984 17.728667038483 0.00475059817701297 -0.710175082739545 5.01107261444136E-23 BNG
ENSG00000137261 23.0116648992577 0.0417311399068192 -0.70626879694329 0.00736742878448766 BNG
ENSG00000113448 38.8888888888889 0.0445643717437885 -0.705491592161748 4.54141174788106E-07 BNG
ENSG00000143126 16.9444444444444 0.0291129750811605 -0.704692138332153 2.74246450392245E-05 BNG
ENSG00000134030 12.3072875559063 0.000780194592826204 -0.703735064599572 0.00423651266925635 BNG
ENSG00000153113 21.9307394036636 0.00374511905818774 -0.703518178232066 1.42459864428974E-14 BNG
ENSG00000179348 24.1163561656628 6.70182429095373E-08 -0.699514617089266 1.00020068482785E-07 BNG
ENSG00000064787 15.4477430029768 0.019433423253655 -0.696607856550066 4.6594488010428E-06 BNG
ENSG00000188026 36.2956810631229 0.0129326007149464 -0.694037324976346 0.023738157713412 BNG
ENSG00000221890 18.7572590011614 0.0308096453721258 -0.690795412733281 0.0176007089136136 BNG
ENSG00000196417 11.4957264957265 0.0125211169053565 -0.690451059239939 0.00048897912138809 BNG
ENSG00000130635 26.9356597600872 0.0271574565142418 -0.685198409708279 1.26290044816402E-26 BNG
ENSG00000111913 30.7936507936508 0.0414896056053475 -0.670840335881557 0.000101763314920898 BNG
ENSG00000150347 14.9859943977591 0.0440225685517479 -0.668489279530708 1.18874074373337E-26 BNG
ENSG00000116711 14.0571532580997 0.0297904753033027 -0.667339028324238 0.0283734297613091 BNG
ENSG00000196227 12.1661315424197 0.0106092170610959 -0.662382727403818 3.58986363598043E-10 BNG
ENSG00000002016 14.5918688937914 0.00312863804951235 -0.655096958010198 0.00272596416552348 BNG
ENSG00000146350 23.3137254901961 0.0146677698628851 -0.653262229789643 0.00944152725963259 BNG
ENSG00000145536 17.1686274509804 0.0427584023858685 -0.64965904943743 2.45479952909829E-05 BNG
ENSG00000158321 27.5966018087147 0.0101861074494865 -0.648265082629678 0.00534070540002623 BNG
ENSG00000171219 12.258064516129 0.00894773991707233 -0.643572286500509 4.99326325232002E-17 BNG
ENSG00000004399 15.4077540106952 0.0417624266596511 -0.643196863834183 0.00186187396969658 BNG
ENSG00000166473 41.9574468085106 0.0183894814151029 -0.637980883020648 0.0350745435774593 BNG
ENSG00000066382 14.5661819919448 0.0154570820713063 -0.63786558271762 0.00347335684687682 BNG
ENSG00000165124 11.3423575860124 0.0380101767330406 -0.63523570720238 0.000592958316482939 BNG
ENSG00000223573 29.0424725656918 6.20881894139791E-09 -0.630849959762384 2.28917372212143E-09 BNG
ENSG00000105357 11.5917067463928 0.0014517892392665 -0.620745655468585 2.7355022608927E-18 BNG
ENSG00000110975 16.7147730254526 0.00872311460033196 -0.620411336725446 0.00614008621027657 BNG
ENSG00000121281 13.0323989807062 0.0342477469741301 -0.620078087582796 0.00134564043630872 BNG
ENSG00000198682 10.4387656702025 0.00204118703000595 -0.618563904087295 1.88074343089455E-10 BNG
ENSG00000188153 11.5046244078502 0.025660119822895 -0.616462780545589 5.88521426071701E-15 BNG
ENSG00000181035 22.9144289294665 0.0147254762161582 -0.613645255425247 0.00752603927024964 BNG
ENSG00000005469 10.3174603174603 0.000499992831155057 -0.609148687022231 3.42678393928905E-06 BNG
ENSG00000075461 15.342989017985 0.00304300349665309 -0.605155649192202 2.28866368636362E-12 BNG
ENSG00000167861 11.7043294290795 0.00136830639884553 -0.600339911716719 1.51057836248098E-06 BNG
ENSG00000104936 10.3047080299292 0.0360010218041492 -0.597332951997686 4.00855123499364E-10 BNG
ENSG00000198691 22.6515151515152 0.0493777709424798 -0.587156714134073 0.000809594934608358 BNG
ENSG00000138336 16.9651741293532 0.0482494140512625 -0.587007426706185 0.000973023962220727 BNG
ENSG00000148180 23.2778604949207 3.57611619264839E-09 -0.58507858029968 1.02616245712461E-21 BNG
ENSG00000164309 18.1738618461881 0.00164648726654438 -0.584962500721158 0.0173490350620861 BNG
ENSG00000165434 12.3711340206186 0.0445444711611503 -0.584708169289755 1.25825478046189E-10 BNG
ENSG00000198846 21.2872559095581 0.0153427691333421 -0.578938713093386 0.00289858288076006 BNG
ENSG00000152284 10.675049351987 0.0459654025511792 -0.573347871818441 0.00169316922396989 BNG
ENSG00000181555 20.5492050978846 0.00757578203996778 -0.57045000701778 0.000633772036520459 BNG
ENSG00000139973 19.6004700352526 0.0305810605288159 -0.570187148040758 0.00480345245979799 BNG
ENSG00000147912 12.7404413500118 0.00303259312325178 -0.569771393690503 0.00602492513846353 BNG
ENSG00000214960 14.1791044776119 0.000599946846525298 -0.566616208240501 0.000191551711513297 BNG
ENSG00000243317 32.6256043725037 0.0311519672052956 -0.56636438224004 0.0104549849382166 BNG
ENSG00000116117 25.4728492983526 0.000960434321705651 -0.565110565625496 0.00619843887025926 BNG
ENSG00000090238 15.1902643933778 0.0213139948134366 -0.556120748810073 0.0493146928859833 BNG
ENSG00000185261 31.9214131218457 0.0314674697603723 -0.553253640993819 0.0362478930997127 BNG
ENSG00000020181 12.4230657780354 0.001966428225144 -0.546427899302171 0.00983148706722227 BNG
ENSG00000128567 18.8532110091743 0.0182896877761265 -0.544534074812149 0.000147772976409115 BNG
ENSG00000120925 12.5353906074451 0.0196933918755241 -0.539247164748559 0.000736410991429054 BNG
ENSG00000197050 18.374307233196 0.000119040562611568 -0.538866086131702 0.00624942651321243 BNG
ENSG00000114423 23.9495798319328 0.0431958123249209 -0.535465534932059 1.7576048201707E-08 BNG
ENSG00000134321 13.6886005560704 0.00997575625153228 -0.522870156352437 7.04810893304969E-05 BNG
ENSG00000241489 24.2486554887694 0.00550099733479635 -0.522263595350611 0.00027164275856533 BNG
ENSG00000137070 12.0733953849137 0.0318905841876695 -0.521952703195358 0.0424631435708416 BNG
ENSG00000166689 24.5501775807919 0.00388073994598053 -0.516848119489151 1.81992158020414E-08 BNG
ENSG00000119686 14.484126984127 0.0332429167482642 -0.51377490313628 0.00213204060595649 BNG
ENSG00000137710 28.6331500392773 0.0389255987511811 -0.509881553601421 1.19794881274545E-11 BNG
ENSG00000128881 14.2607313195549 0.0119137103769155 -0.503303250321294 0.000699512661491251 BNG
ENSG00000127955 41.9444444444444 0.0354275939892162 -0.503216922792406 8.94381266527608E-08 BNG
ENSG00000197321 21.9171814230153 0.036646900840191 -0.501773106156611 5.87968999963141E-08 BNG
ENSG00000268043 22.653156300955 0.00400317217342186 -0.501256784016834 0.0182883160391524 BNG
ENSG00000107099 39.5915678524374 0.0195055406073874 -0.499845887083205 0.00208375996312581 BNG
ENSG00000004468 12.1951219512195 0.038467462223309 -0.498784546838366 2.88191330173453E-19 BNG
ENSG00000083123 22.5 0.0245859854332828 -0.498464583649882 0.000340419237867412 BNG
ENSG00000031003 26.6564318034906 0.0217127474516512 -0.498416665958258 1.62058029177946E-05 BNG
ENSG00000183044 17.5899742930591 0.000384657817382759 -0.497226809344069 8.54488863773884E-05 BNG
ENSG00000124467 23.8176470588235 0.0417943093826968 -0.495318902553343 0.00473377315997699 BNG
ENSG00000177303 25.5813953488372 0.0360479555471996 -0.491974622256619 8.7489021015237E-06 BNG
ENSG00000111961 26.078431372549 0.0407504283176384 -0.491236856643731 2.48976492107798E-14 BNG
ENSG00000185442 23.3378317588844 0.00174367822635544 -0.488240690456756 0.000221616026621544 BNG
ENSG00000010810 14.9159663865546 0.0425652235144658 -0.486449571915371 0.000413551472221232 BNG
ENSG00000111335 19.5384615384615 0.0471474911149716 -0.485212793338396 4.18687964443166E-14 BNG
ENSG00000114770 13.9481413009138 0.028967058065351 -0.484076384162012 4.02271054341108E-08 BNG
ENSG00000137941 11.8190079877553 0.0193456625379411 -0.483014293094851 0.005048594733699 BNG
ENSG00000149260 43.8095238095238 0.0222437844825907 -0.47822513286538 0.0144181754674735 BNG
ENSG00000105492 15.9129901960784 0.0318828061877183 -0.478047296804645 0.00855673971982164 BNG
ENSG00000072858 25.8367856908003 0.0271064512377889 -0.47669201719264 0.0434660969166838 BNG
ENSG00000185630 15.2542372881356 0.0443172927303969 -0.476626525138652 4.68402347442217E-06 BNG
ENSG00000166387 17.7108639863131 0.0295489808875449 -0.475296219553436 0.0011843075479458 BNG
ENSG00000124942 24.7691201681137 0.00103131086256605 -0.474392536230519 3.09603368050903E-14 BNG
ENSG00000008323 37.6282649253731 2.50661784039429E-14 -0.466707226346571 0.00140380214213413 BNG
ENSG00000138303 11.7647058823529 0.00199813755820777 -0.462618563812952 0.000191896181673512 BNG
ENSG00000125266 16.3265306122449 0.0460499274278431 -0.462355578124758 2.7748334758846E-16 BNG
ENSG00000182621 13.279334858637 0.0225777427225306 -0.460113840733944 0.00511236354729573 BNG
ENSG00000126016 16.5492777379367 0.00849881594750249 -0.459114019287442 0.000389295435109945 BNG
ENSG00000107864 12.2122403144301 0.0301314363561356 -0.458141001249102 0.0326818098866569 BNG
ENSG00000136295 13.7074829931973 0.000491213772379682 -0.458073580642192 0.000508698691512146 BNG
ENSG00000171604 27.7815405455329 0.00294096172983806 -0.453648847676855 1.39816502664685E-06 BNG
ENSG00000150556 37.5 0.0109836286899308 -0.453179487183903 0.00172366138067641 BNG
ENSG00000035862 18.5524520315241 0.0351743704365662 -0.451158553185102 4.78852687458916E-18 BNG
ENSG00000137414 10.5544440485652 0.00423248406173437 -0.447689697432434 4.30843512142587E-05 BNG
ENSG00000174456 14.3014604574263 0.0149410499960918 -0.44752315950708 0.03598620057456 BNG
ENSG00000129493 11.0504201680672 0.0144470729314065 -0.446655711835727 0.00778447877637807 BNG
ENSG00000196967 16.2420841567734 0.00025867693432669 -0.445467129297523 0.000224181365577937 BNG
ENSG00000129933 14.3144922384169 0.000432568102183404 -0.444759038098622 0.0374773939660338 BNG
ENSG00000119771 17.2413793103448 0.0388262077612964 -0.44461773449381 0.000411429515895675 BNG
ENSG00000121989 15.3653095370887 0.0302234057665656 -0.444528795988244 1.64215148130686E-05 BNG
ENSG00000118777 19.0268065268065 0.00898381918638679 -0.442408470794975 6.46795535036693E-06 BNG
ENSG00000117114 21.8518518518519 0.0258821518569486 -0.441252757186227 0.000523087175169761 BNG
ENSG00000170365 12.5051419169066 0.00956966372760876 -0.43934074696144 0.00396074346888895 BNG
ENSG00000053254 27.9874213836478 0.040395061954745 -0.439027674185821 1.53454946009585E-08 BNG
ENSG00000115616 12.036010811021 0.0492387190359769 -0.436445306227521 0.000115162802536429 BNG
ENSG00000177694 23.8667282714718 0.00987326807617852 -0.431558175834021 1.29491578718345E-09 BNG
ENSG00000024422 27.6158623984711 0.0168033859609324 -0.430742625234609 0.0292165467312493 BNG
ENSG00000263001 12.9596402622718 0.0185269299019678 -0.427605769403905 1.30049770019362E-15 BNG
ENSG00000104497 10.2733785822021 0.00730893312706585 -0.424642558756677 0.0219969817122393 BNG
ENSG00000076067 10.9556397409263 0.0059602672720826 -0.422942697364189 0.000138197954281027 BNG
ENSG00000111110 16.977466977467 0.000540722238095916 -0.422821207581922 7.20801592208993E-13 BNG
ENSG00000213626 29.9457994579946 0.0377276764664726 -0.414439738739572 9.3078793953285E-09 BNG
ENSG00000172667 23.7246963562753 0.0474848655744828 -0.412851266035975 0.00143250843933229 BNG
ENSG00000105289 15.5648592072388 0.0110678259799135 -0.41174610474983 0.00268329936508764 BNG
ENSG00000135643 14.565610429156 0.0489006771605987 -0.410376121908981 0.0023753800622474 BNG
ENSG00000160325 12.0906136052402 0.0447524194044426 -0.410122008864727 0.0327554626240886 BNG
ENSG00000115904 15.9753456605031 0.00622289236958857 -0.409262861652211 2.21354668124224E-06 BNG
ENSG00000198551 25.9259259259259 0.0263223587684373 -0.408793558121986 0.0144574112408487 BNG
ENSG00000099139 17.3040667923512 0.0260782817068225 -0.408740260817638 2.0345655513523E-11 BNG
ENSG00000076554 30.1587301587302 0.026989458289609 -0.408729640831393 0.000156010695767343 BNG
ENSG00000184384 34.2731829573935 0.0457754145078931 -0.40859384701675 1.90834843581944E-08 BNG
ENSG00000152409 13.0952380952381 0.0089475018307773 -0.406334623724234 1.81980304651701E-06 BNG
ENSG00000185115 14.1691106321331 0.0486297174163698 -0.406285025269877 0.0471767784504365 BNG
ENSG00000133943 17.6787148594378 0.0024986162549337 -0.403301391227878 0.000100742003170586 BNG
ENSG00000011028 23.8768638768639 2.49177149563819E-08 -0.402874720298177 9.42467032388253E-07 BNG
ENSG00000127663 16.3120567375887 0.00199479787932435 -0.401869868853929 2.86929099910416E-05 BNG
ENSG00000275395 40.1215805471125 0.0161209738470805 -0.399832627667512 0.00313125281698433 BNG
ENSG00000140836 22.0262096774194 0.01353803363207 -0.398451891606571 9.35526164448217E-08 BNG
ENSG00000197121 13.3465608465608 0.000216837614926441 -0.398127720768761 6.40513117712682E-05 BNG
ENSG00000147394 14.7713025567546 1.37389673771049E-05 -0.393895478184056 0.00012655160829566 BNG
ENSG00000198624 15.3837342497136 0.0312565681270824 -0.391493449347894 0.019092758640645 BNG
ENSG00000172493 24.3243243243243 0.0326834339365618 -0.38972945200723 0.0234032173352906 BNG
ENSG00000090020 16.4556962025316 0.0416009438220768 -0.388856683389991 0.000518778442434831 BNG
ENSG00000112079 10.1827411167513 0.0100171189668929 -0.388199801705896 1.09379010875715E-07 BNG
ENSG00000138759 36 0.04992950805686 -0.387473859671809 0.0257016002817308 BNG
ENSG00000109323 12.2641509433962 0.0069510470048547 -0.386464027629376 0.00155007758709338 BNG
ENSG00000124006 19.0433862433862 0.0322288314190479 -0.386068098767428 1.87116592734966E-06 BNG
ENSG00000140044 10.7904578132204 9.33890316296523E-09 -0.381947673579412 0.0233492939076747 BNG
ENSG00000067208 10.6948341310447 0.0370695915488687 -0.381634341011633 0.0143609651949742 BNG
ENSG00000149571 15.7046791818436 0.0421796299620546 -0.38124062832878 0.00038268469644075 BNG
ENSG00000166263 15.625 0.00714785082935721 -0.380744321724967 0.000418729852976832 BNG
ENSG00000074621 37.2177055103884 0.0430195524117608 -0.376969458445324 0.0400580722604868 BNG
ENSG00000008300 24.3827160493827 0.00848287737033332 -0.376563351464209 0.0234887642424702 BNG
ENSG00000108370 17.6969178082192 0.000780062895708609 -0.376513232652959 0.0357246871062311 BNG
ENSG00000071909 12.0548116240597 0.0490054769874261 -0.373123857545449 0.0344094454284773 BNG
ENSG00000064655 16.4221364221364 0.018160735094327 -0.370785409945289 0.000286216813915154 BNG
ENSG00000147852 15.3964923393642 0.0338694660141525 -0.370732125655407 8.07912484025131E-06 BNG
ENSG00000103544 44.0711462450593 0.0137093219337725 -0.368911930402233 0.00796346489599808 BNG
ENSG00000142065 21.0619133402863 0.00758559471411506 -0.368867317845923 0.0061016352497874 BNG
ENSG00000166295 22.4340175953079 0.00231052665483842 -0.36854278853606 0.00529971793428787 BNG
ENSG00000196498 27.3656480505796 0.00165568906695835 -0.365364917610353 0.0024253152303974 BNG
ENSG00000107105 12.0634920634921 0.0487293089605174 -0.365260396902254 0.00142528327132613 BNG
ENSG00000110628 11.7507099547164 0.00605011299301495 -0.364197318260097 0.0233052964014367 BNG
ENSG00000145246 10.9188034188034 0.0460646211045671 -0.36064904708903 0.0135260007232632 BNG
ENSG00000164463 17.8451178451178 0.0115418200671948 -0.36011128877525 2.00051018268182E-08 BNG
ENSG00000197852 16.42166344294 0.0328002414526065 -0.358070903678058 0.0463281393522118 BNG
ENSG00000165813 16.8224299065421 0.00315028103681137 -0.35652546997289 8.29764421380416E-05 BNG
ENSG00000048707 14.821083172147 0.0443046839021289 -0.355910194316597 0.00460082542307338 BNG
ENSG00000146411 12.3137254901961 0.0111360967257588 -0.353548977358163 0.000126067031609596 BNG
ENSG00000110987 30.0347222222222 0.0421845647585318 -0.353265409587902 0.000559485941448659 BNG
ENSG00000111271 10.6666666666667 0.008468668423606 -0.353001616041892 0.00560907003355677 BNG
ENSG00000173960 16.7713039566587 0.0483843564132747 -0.352925140306958 0.0394304539177195 BNG
ENSG00000168769 12.5748502994012 0.000650725986752801 -0.352781048803786 0.000226578959147252 BNG
ENSG00000130702 11.1639672292068 0.0372983683188233 -0.35203751427251 2.20623970839814E-08 BNG
ENSG00000183354 12.8326330532213 0.0355388940157917 -0.351864288021866 0.0148775505018936 BNG
ENSG00000166025 11.875 0.00620759228648224 -0.34809950931572 1.15937311348668E-16 BNG
ENSG00000090905 33.4425155584671 0.0422728274876238 -0.347832227062783 0.000471463587356345 BNG
ENSG00000149639 18.4732335027705 0.0195307265727982 -0.347636760659084 0.00218329338042741 BNG
ENSG00000035664 43.3962264150943 0.000533581462920669 -0.347029855464279 0.000251558833382371 BNG
ENSG00000145743 11.3872941770235 0.0352501512573267 -0.345848057083885 0.00115791649628188 BNG
ENSG00000075651 12.3397435897436 0.0465276148281672 -0.345786234742914 1.02844545122384E-06 BNG
ENSG00000173230 10.5312229614822 0.0101047746531274 -0.344274253793976 4.53886715903854E-06 BNG
ENSG00000054267 10.2189781021898 0.0141196697095234 -0.342210031447351 5.14097041637532E-05 BNG
ENSG00000115464 11.1111111111111 0.00133219128075895 -0.340616368337588 0.00164951387603357 BNG
ENSG00000109618 33.179167665149 8.41930404674389E-16 -0.339962017717625 0.00326955308091664 BNG
ENSG00000108389 10.154017148301 0.00553242265274357 -0.33699162338449 0.000125906890521743 BNG
ENSG00000187498 11.733746130031 0.0385511399220718 -0.336870943565742 1.33866133734124E-06 BNG
ENSG00000078114 21.1104684788895 0.0469928272245415 -0.336614795442298 3.31483817017941E-07 BNG
ENSG00000134326 10.0901813475147 0.00955467281308764 -0.335910517196568 0.00768797119644126 BNG
ENSG00000176438 10.1892605633803 0.0159761741102653 -0.334560161543612 0.000737954103415541 BNG
ENSG00000257923 19.1239316239316 1.45731709371131E-05 -0.333068652492565 7.22255658121801E-05 BNG
ENSG00000101871 40.4761904761905 0.0426934459761882 -0.33158031456629 8.68758160318984E-05 BNG
ENSG00000169905 25.7142857142857 0.0267363003963852 -0.331359426161868 5.53490215236054E-14 BNG
ENSG00000187098 19.5980861244019 8.05235237869731E-10 -0.330412292260139 0.00634727754168873 BNG
ENSG00000108840 13.2352941176471 0.000480223775456203 -0.330166204586187 8.37847751568423E-06 BNG
ENSG00000205336 45.4459203036053 0.00752186156988445 -0.326567818510395 0.00273947706529407 BNG
ENSG00000124788 25.7575757575758 0.0428322856458021 -0.3247633955855 5.48725590404673E-18 BNG
ENSG00000138468 19.6808510638298 0.0363634312739796 -0.324452496302909 0.000842061722170376 BNG
ENSG00000119661 10.5009584002487 0.0198710131539813 -0.324146817715437 0.00244892770308636 BNG
ENSG00000158828 22.5434024038254 9.48818804503089E-07 -0.32169317290553 0.000252368264090229 BNG
ENSG00000133059 30.0466355762825 0.0489887123582783 -0.320621897386737 0.00023545527539353 BNG
ENSG00000130723 11.3976750473101 0.000134696004589683 -0.320040985343096 0.000129242633673824 BNG
ENSG00000196352 10.062893081761 0.00946448639676561 -0.319753723653023 0.00103987762735318 BNG
ENSG00000082641 18.8746438746439 0.0413984951095962 -0.319310869004695 3.66328560649142E-11 BNG
ENSG00000213995 14.047976011994 0.0421140380563497 -0.318859345969839 0.00917522191680684 BNG
ENSG00000141736 11.1019736842105 0.015707575378512 -0.318638706904216 1.01759720474176E-05 BNG
ENSG00000196628 18.9379132532125 0.026628993901866 -0.313420129875148 3.28353137619013E-05 BNG
ENSG00000131149 41.1764705882353 0.0493532022723004 -0.312479031368485 8.88086989224702E-05 BNG
ENSG00000178234 11.5294961448808 0.00659775491842036 -0.31233732557587 0.0444075334779666 BNG
ENSG00000168056 19.5390805999268 0.00454352205309947 -0.310082881286277 0.000214762739461813 BNG
ENSG00000033122 29.625590694293 0.00210695325158986 -0.306925931548116 0.00649183678214571 BNG
ENSG00000112304 20.0174825174825 0.0351743704365662 -0.306243573211412 0.0463949995660573 BNG
ENSG00000008083 10.0729517396184 0.0140092913501159 -0.305704155228222 0.00520773838098378 BNG
ENSG00000152104 17.8128950695322 0.0289165706469852 -0.304201627678951 9.3801020593915E-09 BNG
ENSG00000156976 62.4045331362405 1.64036524813892E-26 -0.303223827036908 0.000173375292214423 BNG
ENSG00000148498 18.2381959126145 0.039148232419445 -0.302775645054516 0.000599403897821241 BNG
ENSG00000167972 29.97150997151 0.046054661618327 -0.302562770020427 0.0175183932548747 BNG
ENSG00000109654 22.3880597014925 0.0312572030696368 -0.301685799799022 0.0170642350636622 BNG
ENSG00000107104 10.1918216694849 4.13774880745456E-05 -0.300742764996734 0.00633350140038271 BNG
ENSG00000130844 27.687969924812 0.00390442314567267 -0.300668723482284 0.00719998180056289 BNG
ENSG00000185585 11.5521003818876 0.0392453693308722 -0.299045268650416 7.43191486234675E-05 BNG
ENSG00000217128 12.0838262633091 0.0017609206903326 -0.298728320110066 9.04052804294962E-05 BNG
ENSG00000099260 10.8910891089109 0.0353811040563858 -0.298592047217847 0.000424285345172563 BNG
ENSG00000091157 13.1028368794326 0.0484770918475124 -0.297059220774465 0.0374840228006454 BNG
ENSG00000140416 17.6034134584066 0.000754540256215715 -0.296072693115342 7.8888780545974E-08 BNG
ENSG00000088280 13.6121812511466 0.0043559525569461 -0.295958148559171 3.52360850016329E-05 BNG
ENSG00000196267 18.834768250973 0.0114164686143015 -0.295920444805625 0.0101731668155433 BNG
ENSG00000204217 24.3243243243243 0.00155707895508587 -0.294392145198983 8.00814045879748E-08 BNG
ENSG00000118816 17.7777777777778 0.0459158478090898 -0.293827410959701 0.000793611995264555 BNG
ENSG00000146648 21.6057812677819 0.0164588521031709 -0.293703786413115 2.29989877751664E-08 BNG
ENSG00000075568 14.4736842105263 0.028405831268229 -0.293035982058586 0.0302709234446858 BNG
ENSG00000105426 23.3977172958736 0.00158843839337624 -0.29136312889229 9.61011029442332E-06 BNG
ENSG00000142784 12.641432367221 2.75713514771609E-08 -0.291309926911496 0.000705471601159669 BNG
ENSG00000115468 24.9363327674024 0.012367393390153 -0.290583721544564 0.0401381507632302 BNG
ENSG00000119912 14.340675671213 0.0223871948498093 -0.288796061228694 0.0179505502465591 BNG
ENSG00000214655 13.1659836065574 0.0357874542093045 -0.288774708212451 0.00266065262221928 BNG
ENSG00000162825 14.2034333025851 0.00666121923321421 -0.286100015751736 0.0198036139611593 BNG
ENSG00000133424 39.5833333333333 0.0244052415164296 -0.285218427692496 0.0188359404674102 BNG
ENSG00000184677 27.8225806451613 0.0381796105921428 -0.285200061636264 0.0330078205932338 BNG
ENSG00000116005 31.1556603773585 2.10459449932301E-07 -0.28407646921438 0.000921125149288969 BNG
ENSG00000165240 10.3479853479854 0.011765225239976 -0.28314719196807 7.80285429525471E-09 BNG
ENSG00000133983 39.5604395604396 0.0414994516121064 -0.28200331050987 0.0264347269628561 BNG
ENSG00000151553 18.560074801309 0.0421356350946662 -0.280613980013202 0.00535868165740882 BNG
ENSG00000186448 20.9177489177489 0.000190407930326322 -0.279901421655579 0.0243344047039009 BNG
ENSG00000139266 11.5023275497249 0.0251514349771169 -0.277918279325949 0.0404238161915395 BNG
ENSG00000164855 17.4666035744185 0.00378786634448218 -0.268774379016911 0.0101022767524733 BNG
ENSG00000054965 28.4817875726967 0.0118905351512364 -0.267963637356679 0.000293100155619997 BNG
ENSG00000148842 28.0701754385965 0.0148914319157164 -0.267695783203657 0.00377595052802898 BNG
ENSG00000123636 12.257053291536 0.0303309564762876 -0.267416852462943 0.011351823485927 BNG
ENSG00000115170 12.5139206377117 0.0109195293972354 -0.267382852871544 0.0370125180568233 BNG
ENSG00000133639 18.9965359258018 0.0137735837044225 -0.267168024889054 0.0028996243944354 BNG
ENSG00000126858 19.9737532808399 0.0103436202041154 -0.266499681624815 0.0311298311886331 BNG
ENSG00000112855 11.1111111111111 0.0274407754224659 -0.264771736543482 0.0349592186244644 BNG
ENSG00000162434 11.1013986013986 0.0400147705940586 -0.263612329451374 4.94379178207903E-08 BNG
ENSG00000140379 -13.0602240896359 0.0419869667954231 7.75933340719466 0.0367406623189661 BNG
ENSG00000102445 -22.4608590927338 0.0136717844177114 7.1632303488683 0.0201523496528516 BNG
ENSG00000198626 -30.4647160068847 0.0317766894353491 5.4374053123073 0.0297721172788166 BNG
ENSG00000273217 -11.6228070175439 0.0495363996427847 5.32192809488736 0.0445013525732622 BNG
ENSG00000186603 -10.4218502246818 1.74723393165367E-06 4.88264304936184 0.00562268869170042 BNG
ENSG00000167772 -20.6896551724138 0.026213853591628 2.89710009241571 3.24861125698761E-10 BNG
ENSG00000288520 -18.5674522845575 0.0293128390705597 2.7004397181411 0.0288626152504842 BNG
ENSG00000152953 -25.4347826086957 0.0389556921466233 2.32192809488736 0.0352806015308715 BNG
ENSG00000163359 -18.6857721812129 0.00839883744965913 2 0.0453917622597636 BNG
ENSG00000197641 -11.5761478163494 0.00503948227834267 1.92619869958988 1.92719648884193E-25 BNG
ENSG00000205403 -15.8402203856749 0.0062625386976592 1.68182403997374 0.0124171144084143 BNG
ENSG00000138675 -13.9705882352941 0.00218543164097531 1.58496250072116 0.00663087291080351 BNG
ENSG00000178726 -11.8489223129544 0.00199516551116929 1.52281005197375 2.48269726859935E-30 BNG
ENSG00000154678 -19.3846556690233 0.0433527558360389 1.39854937649027 0.0282987501155144 BNG
ENSG00000130164 -11.0260942983035 0.0115934255677052 1.38476472870143 1.68437434910057E-68 BNG
ENSG00000138944 -37.8117457763475 0.0169445660029793 1.37586690199816 0.00219311995651581 BNG
ENSG00000197646 -12.0122574055158 0.0251916789947428 1.36950657725287 4.76360889303288E-20 BNG
ENSG00000163485 -17.520151730678 0.00250341105617622 1.34513548604869 0.0424124417976442 BNG
ENSG00000188001 -21.5264586160109 0.00189270004090834 1.33763299559323 1.51293275212706E-05 BNG
ENSG00000285816 -15.6369183829138 0.0239049278657468 1.29972421516298 1.68350938440851E-07 BNG
ENSG00000177374 -21.5589376667475 0.00445208163839317 1.24285652389617 0.0138731645825835 BNG
ENSG00000090339 -10.6666666666667 0.0410133958281216 1.22517486200778 1.17341214937116E-27 BNG
ENSG00000104368 -34.1463414634146 0.0182409938245087 1.1794767156618 4.32168441024132E-39 BNG
ENSG00000169991 -17.1866447728517 0.0158204811842248 1.15497288407955 1.9806121954697E-32 BNG
ENSG00000175505 -27.9661016949153 1.06348633808996E-05 1.13024667675028 7.38979815808529E-11 BNG
ENSG00000196639 -15.4970760233918 0.00090947165898245 1.12687176584051 2.79229839886979E-11 BNG
ENSG00000196083 -36.3636363636364 0.000249970412403766 1.1188160178939 1.91714465012516E-34 BNG
ENSG00000107957 -18.655090765588 0.043587140391366 1.10378045790299 5.22642208471637E-05 BNG
ENSG00000167914 -19.7192513368984 0.00810848185643203 1.0790715709912 0.00212095639712 BNG
ENSG00000124496 -11.9527769853635 0.00548776059044123 1.05475976867859 9.74227997345186E-09 BNG
ENSG00000134107 -20.5533596837945 0.0102286108939419 1.01740704590947 5.59821544185794E-34 BNG
ENSG00000112096 -15.7002858309514 0.0276978683819853 1.00829655641631 0.0277179645543597 BNG
ENSG00000136026 -20.8110516934046 5.06625982084557E-06 0.981140972748685 0.000121641120032545 BNG
ENSG00000137936 -11.6054875787913 0.0387043048384392 0.940973373349762 1.93812440346207E-27 BNG
ENSG00000125730 -12.3979591836735 0.000755350080859917 0.940197017497937 1.10578488599274E-13 BNG
ENSG00000142634 -25.2007557864903 0.0255231120152627 0.908622445824029 4.92613125327356E-49 BNG
ENSG00000182326 -12.3648537401899 0.0273998846984687 0.878141657126903 3.79553006148258E-05 BNG
ENSG00000111700 -51.4193548387097 0.00599089375902751 0.867939147909315 0.0025682878525908 BNG
ENSG00000129521 -35.8974358974359 0.00643442018997199 0.84358980503217 2.59806804251617E-05 BNG
ENSG00000075131 -13.2420091324201 0.0289563274100958 0.816530118457942 0.00474497942871165 BNG
ENSG00000114541 -14.0845070422535 0.034354834947395 0.815747086313056 1.42922170794235E-22 BNG
ENSG00000127507 -23.5303617571059 0.00980477148159086 0.810338780496422 0.000547643416920011 BNG
ENSG00000172927 -16.2748744136993 0.0160248077870733 0.808903709153095 0.00348914353770542 BNG
ENSG00000174640 -20.5257054503286 0.029565719454343 0.800012946716431 0.00209466071836222 BNG
ENSG00000156299 -17.5735294117647 0.0277298044419221 0.792603966282472 5.65041318074885E-06 BNG
ENSG00000088727 -18.2795698924731 0.0496766885347703 0.787318886643381 0.0218939732192762 BNG
ENSG00000105137 -11.5606936416185 0.00939974597671166 0.786429083203057 0.000279118337134963 BNG
ENSG00000128965 -18.6904761904762 0.000282827758537118 0.782165860625856 1.505866464105E-06 BNG
ENSG00000103546 -11.6170689719897 0.0154089401089322 0.758806642705791 0.0418443211370969 BNG
ENSG00000123358 -25.8726647000983 0.0130489763385992 0.737561626003184 5.9249188956385E-06 BNG
ENSG00000138829 -31.6725352112676 0.0372853032268371 0.713477241243836 0.0417689085213096 BNG
ENSG00000102743 -18.4110885733604 0.00457691456434629 0.707392478782857 0.00209427175210604 BNG
ENSG00000157557 -22.9957805907173 0.017203962372102 0.694034680852988 1.74996558874179E-33 BNG
ENSG00000034152 -18.8859520313265 0.0167888007588856 0.693086631049928 5.24816371136761E-05 BNG
ENSG00000213397 -27.1225071225071 0.00434588003696628 0.689932060322699 0.000404367736333311 BNG
ENSG00000131746 -10.5288590604027 0.0264465832690492 0.678937554055473 5.60206611487306E-12 BNG
ENSG00000165799 -24.1837299391256 0.00857990103345192 0.678071905112639 0.0177003751106127 BNG
ENSG00000142089 -11.331290432414 5.65365659019406E-05 0.673593087060183 1.29781289919547E-25 BNG
ENSG00000184408 -38.2692307692308 9.49729224589839E-06 0.667424660913128 0.0384781428522723 BNG
ENSG00000126091 -21.6867469879518 0.00889633435336452 0.626850897011178 0.00839083915114877 BNG
ENSG00000143322 -26.9976498237368 0.000254774068165335 0.617742991222575 1.13702174725912E-26 BNG
ENSG00000127666 -13.0050658561297 0.00325853898090201 0.617666665793861 2.82406615980706E-06 BNG
ENSG00000154217 -23.3516483516484 0.0493895123654999 0.615899482470343 0.000662697989781469 BNG
ENSG00000112159 -23.3950694531348 0.0108221249561433 0.610708775591004 0.00592967658047154 BNG
ENSG00000104419 -23.7073170731707 0.049221174709303 0.609880299095611 1.42913107270399E-30 BNG
ENSG00000182010 -17.0862470862471 0.0355002358593476 0.597418933145846 1.36143320589088E-07 BNG
ENSG00000107937 -16.5659543109801 2.71661545092223E-06 0.597234370126258 1.09995147692887E-07 BNG
ENSG00000119681 -19.1963826944815 0.00719070703708989 0.595560346860678 1.84681312504672E-05 BNG
ENSG00000128294 -29.5713229859571 0.000142543647709696 0.59321592735501 0.0345827448979271 BNG
ENSG00000154655 -33.4452064789143 0.00390173354630116 0.59183345241 1.03930323127386E-06 BNG
ENSG00000165650 -16.3934426229508 0.0378421421189818 0.579453598291972 0.0453755163033109 BNG
ENSG00000140945 -54.1404612159329 6.28268106238419E-05 0.574978412148533 0.00366332390862918 BNG
ENSG00000131941 -10.5882516781327 0.0184141127143809 0.573258396588686 1.74407845148977E-14 BNG
ENSG00000136492 -11.7223083063342 0.0117767722940519 0.572251448971601 2.26692368906575E-07 BNG
ENSG00000166444 -42.5475687103594 0.0295866997565394 0.56545984417481 4.99627819602438E-09 BNG
ENSG00000156113 -17.9012547914687 0.0127279978159161 0.562791119722535 0.0227562050853387 BNG
ENSG00000159110 -14.3515151515151 0.00553207872317444 0.562541206013363 0.0121492862435441 BNG
ENSG00000113318 -17.8626289225368 0.00157714237650006 0.562313761775957 0.000273593476721578 BNG
ENSG00000142185 -42.8235294117647 0.0245087649772323 0.561438218660123 0.0293332076847108 BNG
ENSG00000050820 -20.3125 0.0271861387486728 0.556139137843372 4.24342943708694E-08 BNG
ENSG00000115641 -24.035964035964 0.0339725594017967 0.552391837873705 1.0526762753976E-29 BNG
ENSG00000060558 -13.6 0.00601386696065428 0.550616755318472 0.00025669154956774 BNG
ENSG00000147065 -19.4475138121547 0.0241049580541904 0.53654301642417 8.11287354734939E-30 BNG
ENSG00000160214 -12.9454545454545 0.0390164434434383 0.535814851392069 1.07390707704074E-05 BNG
ENSG00000162419 -12.7713474557764 0.0319921945269321 0.525155335489124 0.00223599875365847 BNG
ENSG00000167670 -11.6883116883117 0.0192095726179785 0.521039303621415 0.000147383634918683 BNG
ENSG00000171488 -12.2956730769231 0.0172368472444858 0.513239813177653 0.00114332064975316 BNG
ENSG00000073712 -16.9811320754717 0.0408786639650407 0.512037234330269 1.28218578484813E-07 BNG
ENSG00000196792 -31.4427103159497 0.000199788774540287 0.507954281998884 0.0461085059867358 BNG
ENSG00000050165 -16 0.049872192771084 0.507308499457133 1.72938165941866E-06 BNG
ENSG00000171453 -13.2422402159244 0.0269713990389909 0.50397390606564 1.80289167441687E-05 BNG
ENSG00000147689 -10.2777205394143 0.0296131818152576 0.498448488864361 2.63996760589321E-05 BNG
ENSG00000134686 -11.2458193979933 0.0490807313383056 0.49406131874452 5.10831089048732E-11 BNG
ENSG00000065534 -26.1363636363636 4.58581903515231E-05 0.492937258882307 0.00489440648365668 BNG
ENSG00000123130 -31.9319319319319 0.0018567584274336 0.489750774803252 0.00176615938851862 BNG
ENSG00000275740 -15.503581963759 0.0453593619314214 0.487931412162827 0.00587654338965406 BNG
ENSG00000156398 -18.8524590163934 0.000348784129373148 0.487008729785849 0.0128476275418884 BNG
ENSG00000285901 -12.202502379981 0.00468928631103516 0.485665901665924 0.00265331917593365 BNG
ENSG00000039560 -12.9437229437229 0.0263720785924747 0.483171930628531 6.66514055683011E-11 BNG
ENSG00000100023 -22.8361871673974 0.00133219128075895 0.482668327207678 0.0105207229595894 BNG
ENSG00000182484 -20.2096926023306 0.0481433701401008 0.48255939531705 0.0492338919447914 BNG
ENSG00000132334 -10.0267752552016 0.0242987932391369 0.475178385333266 7.6377217442228E-05 BNG
ENSG00000083454 -14.9709934735315 0.0364912699901172 0.474966356535059 0.036003407428261 BNG
ENSG00000076003 -11.8291944276196 0.0218100885442315 0.473426901781583 1.34801366061123E-12 BNG
ENSG00000177706 -13.3480709381021 2.24772238051684E-13 0.471523213919535 5.28191185180264E-07 BNG
ENSG00000100065 -20.1939115929942 0.0130722284127865 0.470268230654617 3.15996374866782E-08 BNG
ENSG00000173530 -21.7687985014718 2.8140664777479E-05 0.468398314663583 0.000143462486375593 BNG
ENSG00000137478 -24.3654822335025 1.64835961175456E-10 0.46837326134002 0.00725447648208387 BNG
ENSG00000171132 -21.3210296260321 0.0256886794368254 0.468248157279246 0.0072417886941382 BNG
ENSG00000149177 -10.7919678714859 0.00278325904974516 0.467617414400212 0.0446129236346782 BNG
ENSG00000185215 -22.6086956521739 0.0223192198423381 0.466484134892728 5.69006853725303E-12 BNG
ENSG00000103187 -11.2068965517241 0.0387775128062209 0.46390663278 2.51230178927757E-17 BNG
ENSG00000134013 -18.2029598308668 0.0190547132705856 0.462967204381542 4.76544917479903E-12 BNG
ENSG00000141682 -10.1941747572816 0.00214392520301642 0.462655005004157 1.81835401657973E-05 BNG
ENSG00000134775 -15.8536585365854 0.0272829985423177 0.459090918425327 3.0528769923768E-05 BNG
ENSG00000188677 -24.9844564541585 0.000168636053320139 0.45812961249548 0.0430808758707585 BNG
ENSG00000240053 -12.7484296158995 0.0296606338382643 0.455843715666729 0.0294401639525279 BNG
ENSG00000160799 -40.3155818540434 1.69713921674551E-06 0.452833529552961 0.00501376556672779 BNG
ENSG00000109861 -12.7042483660131 0.0431789067221677 0.448219439704906 4.16593315448456E-10 BNG
ENSG00000157551 -12.9955514265807 0.00753561613606589 0.447594054452798 0.00230304213579688 BNG
ENSG00000197498 -13.6572622779519 0.0123223125411247 0.447023051589182 0.00184985462302071 BNG
ENSG00000196776 -11.6533425279246 0.00849214239415063 0.437581426271295 0.000188816039026614 BNG
ENSG00000197417 -19.751056964842 4.01914267959484E-11 0.432638130125041 0.0169974449289784 BNG
ENSG00000092969 -12.7301640441915 0.0319446080797506 0.432025567700634 1.3965214654066E-08 BNG
ENSG00000148459 -31.8840579710145 0.00568478148180565 0.429364974832301 0.0433078449692255 BNG
ENSG00000162433 -11.2694811599921 0.00173237839008872 0.425793896102561 4.5580052209163E-08 BNG
ENSG00000101546 -10.7843137254902 0.0418187681953032 0.423722096644087 0.00495092158758691 BNG
ENSG00000120254 -11.3635157369566 0.0402595578220733 0.416225112127262 3.11356337626985E-05 BNG
ENSG00000165689 -12.2080440996325 1.88001695122534E-05 0.414491747412722 2.01925319811362E-07 BNG
ENSG00000166949 -12.8427912028238 0.0166308090053364 0.410357367727777 6.43088240364512E-15 BNG
ENSG00000139343 -16.9364413656437 0.00648669118839666 0.408633088879767 0.000641235169794379 BNG
ENSG00000166068 -13.5135135135135 0.0351743704365662 0.40489548885823 0.000120660653415139 BNG
ENSG00000105928 -11.1111111111111 0.035093062877173 0.40377048746052 4.22656711665809E-06 BNG
ENSG00000141076 -14.051533802869 0.0280107819608233 0.401002142074746 4.02203339977718E-15 BNG
ENSG00000156787 -29.8706240487062 0.0468147702659847 0.395736943319907 4.74318720256828E-05 BNG
ENSG00000170379 -32.4734249416645 0.0170665902919789 0.394768901772857 0.0271338669211037 BNG
ENSG00000146232 -14.0488110137672 0.00474973349274566 0.392149770861626 0.00332674089463065 BNG
ENSG00000079337 -14.4724556489262 0.0405054892640396 0.390891411947141 0.0064912127527941 BNG
ENSG00000113356 -10.45280468574 0.0174053941207476 0.390375445044573 0.0389512379357209 BNG
ENSG00000126602 -10.4268292682927 0.00276913810072556 0.38577551135716 1.82359994148375E-09 BNG
ENSG00000132470 -11.2192569335427 0.00310423617780924 0.383379173420695 4.30403166435463E-06 BNG
ENSG00000012048 -16.2792058516196 2.78883070497941E-05 0.381249185822562 2.9090055172466E-05 BNG
ENSG00000173744 -14.4028520499109 0.000202174864163991 0.379563150298012 0.00111750633730919 BNG
ENSG00000276043 -20.8995584988963 0.0126091804239962 0.377966627324228 1.13351188388114E-08 BNG
ENSG00000104998 -31.9047619047619 0.00809423555363895 0.377373601776779 0.00378313393633686 BNG
ENSG00000020426 -13.6842105263158 0.0110742001662289 0.372042127924171 0.0012578498521631 BNG
ENSG00000109519 -11.2951807228916 0.0396862905145413 0.37164683450387 0.000248352981138557 BNG
ENSG00000117411 -17.4176805078022 0.0164899944153455 0.370426330875051 5.04544077240487E-06 BNG
ENSG00000072110 -21.177245601798 0.00391150829143861 0.36814862841075 2.13959571937118E-10 BNG
ENSG00000143324 -18.1824882075472 9.45950789324614E-06 0.366843989575849 3.25957742537955E-05 BNG
ENSG00000174501 -20.0966962127317 0.0310230617351151 0.36513259345253 0.00490678291116583 BNG
ENSG00000132434 -27.9419609516697 0.0393749617732968 0.363383323510468 0.0187163147277958 BNG
ENSG00000185344 -27.9569892473118 3.86317235533345E-06 0.361820822010875 0.0321913603940228 BNG
ENSG00000198742 -16.2399969659043 0.00501008174211403 0.360277274531718 0.000538983577484304 BNG
ENSG00000151693 -25.9456333140544 2.19912826474313E-05 0.354638036216122 4.57279940276897E-06 BNG
ENSG00000156795 -38.0377358490566 0.0144869300714228 0.343348146097523 0.00551779589047665 BNG
ENSG00000077150 -12.6892461370376 0.0306893423114589 0.3412428507965 3.37086858397517E-05 BNG
ENSG00000108021 -22.2047389855609 0.00106602646373337 0.339704174053552 0.000135551547207724 BNG
ENSG00000119408 -61.0294117647059 2.8440811599222E-06 0.338972363751516 0.00265932587383534 BNG
ENSG00000197063 -15.2279763580073 0.00842915807812787 0.334400889337693 5.31922911707779E-05 BNG
ENSG00000180730 -20.8983251564879 7.90954011912372E-07 0.333836788722445 0.00201645424663414 BNG
ENSG00000111252 -11.7093023255814 0.00998741143562405 0.33125115813949 3.52825565234949E-05 BNG
ENSG00000145819 -10.7178340784898 0.0396605018365575 0.330677158225405 0.00536155887245511 BNG
ENSG00000196535 -11.4600972797694 0.0161093684855209 0.328931310926499 0.0123758316344305 BNG
ENSG00000162613 -17.3644474948524 0.0324067530449809 0.328447709136984 3.13022134509149E-06 BNG
ENSG00000062038 -13.4920634920635 0.0238922126161395 0.328215850647634 2.00014818397733E-10 BNG
ENSG00000092853 -15.9663865546218 0.0125512798926505 0.327634213232578 0.000642703698649307 BNG
ENSG00000092470 -29.1854072963518 0.0296202538605799 0.326458938833974 0.0020964526533321 BNG
ENSG00000186314 -17.1071438989528 0.0231358749611893 0.326345262946965 0.0329328808988499 BNG
ENSG00000166166 -10.68524970964 0.0192164922708738 0.325728283849324 0.0136212340309268 BNG
ENSG00000196550 -12.6049933383537 0.013463315146511 0.323951510423659 0.00514137736104517 BNG
ENSG00000104852 -10.1283636828863 0.0139631200255514 0.320980373575367 0.0272299406051771 BNG
ENSG00000165304 -18.1269162872805 0.000643598541175108 0.319129648728101 2.3370565052397E-05 BNG
ENSG00000214113 -12.5010029687876 0.0156887342030493 0.31875051786639 0.0469611212957108 BNG
ENSG00000138442 -13.8278388278388 0.0213187887833145 0.318574659777552 4.27600892666218E-05 BNG
ENSG00000112972 -16.1152949614488 0.0126838220569589 0.315001497982013 0.000188738979456314 BNG
ENSG00000169180 -13.2955936352509 0.000849752872498409 0.314632885879975 0.00135627094179664 BNG
ENSG00000049541 -12.280701754386 0.0169546622797692 0.31359405800916 0.0156478701318847 BNG
ENSG00000132676 -11.035419452049 0.0245033065121283 0.312063598061888 0.000208407937187256 BNG
ENSG00000141526 -23.1404958677686 0.00261240939848165 0.311619739598562 0.000591786040639583 BNG
ENSG00000126698 -25.1425106124924 0.00166587580328008 0.306617549211211 5.18237372697286E-05 BNG
ENSG00000114480 -10.9884201819686 0.0223217340619718 0.306344199668908 0.00604581620290016 BNG
ENSG00000120539 -19.3901132968829 0.00343396625615726 0.305597893946784 0.00455858021237978 BNG
ENSG00000105486 -15.5844155844156 0.0453494958684685 0.30478296776648 0.0373791119128896 BNG
ENSG00000131620 -13.3653830413027 0.0252948591576672 0.303751434937819 0.00347391075687533 BNG
ENSG00000254087 -10.2983725135624 0.0488287796275528 0.300166838538387 3.02913127470451E-07 BNG
ENSG00000035403 -11.7315415508187 0.0441497518533752 0.299008368980096 6.87026731676217E-09 BNG
ENSG00000083635 -19.9529373580006 0.0381200922165914 0.29870686861847 0.0119954298989839 BNG
ENSG00000004897 -11.1111111111111 0.00478915076025825 0.295393259079194 0.00466842591071524 BNG
ENSG00000050344 -10.0816582914573 0.015707575378512 0.293012352905917 1.17008748072877E-05 BNG
ENSG00000153179 -21.377606263361 0.0203578875585219 0.292518165356911 7.62622000548776E-07 BNG
ENSG00000160957 -23.3835005574136 0.00101322585276091 0.290326906767959 0.0265100950636294 BNG
ENSG00000163808 -18.9340915718868 0.00301633219510026 0.289017609465231 0.0494132444071175 BNG
ENSG00000178409 -12.9095116307393 0.000364087446723097 0.2883218952718 0.00811924789522798 BNG
ENSG00000175054 -11.7296606644084 0.00106320574342346 0.287648371836303 0.0146795244446537 BNG
ENSG00000173039 -27.7398958591619 0.00656136475063831 0.285633241047627 0.0231883773624121 BNG
ENSG00000150753 -14.4251968503937 0.0218407491899384 0.284904189377523 1.84163220481641E-11 BNG
ENSG00000107130 -14.7435897435897 0.00549774755816806 0.281455174998768 0.000936775496395401 BNG
ENSG00000100749 -18.5744810744811 0.0250788833752389 0.280627114774547 0.00344286194449417 BNG
ENSG00000183856 -13.0434782608696 0.0401499412717785 0.279732061639544 5.33521552830561E-05 BNG
ENSG00000175792 -10.6004901960784 0.00203845771080645 0.279137832001109 0.000439440796344355 BNG
ENSG00000074201 -24.7899159663866 0.0403083767489215 0.278172389751471 0.00542221796366029 BNG
ENSG00000184900 -30.8723265153638 0.000377128553514499 0.273174201869466 0.000220867190177533 BNG
ENSG00000105220 -18.7007110536522 0.00212214059295916 0.272702090740145 0.00812206968916581 BNG
ENSG00000163293 -10.8718487394958 0.0497617848609876 0.272023189061049 0.0270981163833197 BNG
ENSG00000131378 -41.025641025641 0.00822190406098067 0.271778685197926 0.0160680013219797 BNG
ENSG00000001497 -16.4869398514258 0.0137608208710987 0.268705941554237 0.00102904917257424 BNG
ENSG00000135932 -13.8342133248755 0.000662445859206366 0.267400192087818 4.11766224712256E-06 BNG
ENSG00000285077 -15.7713467817686 0.045902210404969 0.266423004081944 0.0468961829118312 BNG
ENSG00000156873 -12.9756450446106 0.000127244227100649 0.265336666731864 0.031407509745278 BNG
ENSG00000182022 -14.7592262313075 0.00110428414136549 0.26444863953694 0.000757398141982721 BNG

2.5. MSCs promoting the demethylation of the transcription factor NGF and then upregulating KRT17 and FOXJ1

We predicted the transcription factor binding sequences of 66 genes whose methylation levels were negatively correlated with transcription levels in the promoter region and found that 9 genes were rich in transcription factor binding sequences (Table 7). Based on the gene expression correlation and sequence analyses, we selected 4 transcription factors to construct a map of the transcription factor and target gene interactions of MSCs to reverse TEC senescence via Cytoscape (Fig. 5A). In this network, NGF is the central regulatory factor, targeting 108 genes, including many antiaging-related motifs, such as KRT17, FOXJ1. The expression level of NGF was positively correlated with that of KRT17 and FOXJ1 (Pearson's r = 0.92 and 0.74, Fig. 5B). Indicating that promoter demethylation is closely related to the increase in mRNA expression during MSCs reversal of TEC ageing.

Table 7.

Transcription factors correlate with target genes.

TF Gene Score
ELL2 TRIM7 4.465
ELL2 TRIM41 4.317
ELL2 HMGN4 3.913
ELL2 BORCS6 3.857
ELL2 ALOXE3 3.803
ELL2 ZNF184 3.755
ELL2 RNF207 3.587
ELL2 HES7 3.579
ELL2 H4C12 3.578
ELL2 AURKB 3.566
ELL2 UBC 3.538
ELL2 H4C8 3.514
ELL2 SDR39U1 3.409
ELL2 B4GALT3 3.336
ELL2 GBA 3.3
ELL2 SHF 3.265
ELL2 HARS2 3.246
ELL2 HARS1 3.242
ELL2 PPOX 3.235
ELL2 USP21 3.14
ELL2 PQBP1 3.138
ELL2 H4C11 3.132
ELL2 ZMAT2 3.117
ELL2 H2AC11 3.113
ELL2 SRCIN1 3.098
ELL2 H2BC11 3.097
ELL2 TIMM17B 3.093
ELL2 ANG 3.084
ELL2 RPL22 3.082
ELL2 H2BC17 3.079
ELL2 CCDC77 3.075
ELL2 H2AC17 3.063
ELL2 H4C9 3.009
ELL2 RNASE4 3.003
ELL2 ZNF335 3.002
ELL2 HOXA5 2.98
ELL2 WDR13 2.958
ELL2 S100A13 2.946
ELL2 BDP1 2.942
ELL2 S100A1 2.935
ELL2 PIN1 2.91
ELL2 H2AC15 2.884
ELL2 HBP1 2.875
ELL2 LDHA 2.875
ELL2 LOC105371063 2.872
ELL2 BAZ2A 2.865
ELL2 CALM1 2.861
ELL2 DNAJA3 2.858
ELL2 LOC107985657 2.853
ELL2 BRMS1 2.844
ELL2 CCDC130 2.842
ELL2 H2BC15 2.837
ELL2 TMEM107 2.836
ELL2 KHK 2.821
ELL2 ZBED9 2.818
ELL2 UFC1 2.793
ELL2 CSKMT 2.774
ELL2 UBL5 2.772
ELL2 C11orf98 2.767
ELL2 H2BC9 2.748
ELL2 TMEM205 2.738
ELL2 TCEANC 2.733
ELL2 RACK1 2.733
ELL2 H3C7 2.733
ELL2 DPP9 2.732
ELL2 B4GAT1 2.717
ELL2 FAM189B 2.714
ELL2 RIN1 2.708
ELL2 CCDC159 2.707
ELL2 LBHD1 2.703
ELL2 UQCC3 2.7
ELL2 SLC25A35 2.699
ELL2 GCDH 2.685
ELL2 ZNF775 2.685
ELL2 DISP3 2.682
ELL2 ALG3 2.679
ELL2 CORO7 2.675
ELL2 CORO7-PAM16 2.675
ELL2 WBP1 2.674
ELL2 NOD1 2.671
ELL2 MUC1 2.663
ELL2 NCOA3 2.661
ELL2 EEF1AKMT4 2.657
ELL2 EEF1AKMT4-ECE2 2.657
ELL2 H3C8 2.655
ELL2 INO80B 2.643
ELL2 QRICH1 2.639
ELL2 INTS5 2.636
ELL2 BSDC1 2.635
ELL2 TP53I3 2.633
ELL2 BSG 2.632
ELL2 IER3 2.631
ELL2 JUP 2.629
ELL2 H3C12 2.624
ELL2 H2BC10 2.624
ELL2 SIRT4 2.616
ELL2 SCAMP3 2.609
ELL2 PSAP 2.608
ELL2 HSPA6 2.607
ELL2 H4C2 2.605
F2R MTRNR2L1 0.888
F2R COL23A1 0.727
F2R LOC105378947 0.482
F2R TMEM242 0.481
F2R ARHGEF18 0.464
F2R LOC101060017 0.244
F2R PEX11G 0.07
F2R CLK4 0.057
F2R OR4F16 0.046
F2R TEX45 0.039
F2R MMEL1 0.029
F2R POTEA 0.026
F2R FRG2B 0.013
F2R ZNF358 0.011
F2R ZDHHC14 0.009
F2R MCOLN1 0.007
F2R PNPLA6 0.003
F2R PRXL2B 0.001
F2R HGSNAT 0.001
F2R LOC107984841 0.001
F2R TTC34 0.001
GMEB2 UBE2M 3.861
GMEB2 CHMP2A 3.676
GMEB2 TAF6 3.455
GMEB2 CNPY4 3.448
GMEB2 MCM7 3.444
GMEB2 AP4M1 3.442
GMEB2 SRSF2 3.395
GMEB2 MFSD11 3.394
GMEB2 VPS52 3.39
GMEB2 RPS18 3.39
GMEB2 ZNF517 3.264
GMEB2 B3GALT4 3.233
GMEB2 RPL8 3.126
GMEB2 MOGS 3.116
GMEB2 WBP1 3.081
GMEB2 MZF1 3.074
GMEB2 NAA80 3.062
GMEB2 HYAL3 3.062
GMEB2 HYOU1 2.977
GMEB2 LIN37 2.974
GMEB2 PROSER3 2.974
GMEB2 WDR74 2.969
GMEB2 TASOR 2.929
GMEB2 HSPB6 2.92
GMEB2 TAF12 2.911
GMEB2 YIF1B 2.909
GMEB2 MRPL53 2.894
GMEB2 C19orf33 2.879
GMEB2 NAA38 2.872
GMEB2 USP21 2.871
GMEB2 INO80B 2.87
GMEB2 COPS6 2.866
GMEB2 CYB5D1 2.865
GMEB2 MBLAC1 2.855
GMEB2 DBP 2.842
GMEB2 PSENEN 2.84
GMEB2 TUSC2 2.835
GMEB2 U2AF1L4 2.824
GMEB2 SEC22C 2.814
GMEB2 GLTPD2 2.804
GMEB2 CKS1B 2.803
GMEB2 TRIM28 2.796
GMEB2 SHC1 2.793
GMEB2 KCNK6 2.786
GMEB2 CALR 2.786
GMEB2 DHPS 2.777
GMEB2 RAD23A 2.776
GMEB2 SS18L2 2.776
GMEB2 TMEM88 2.764
GMEB2 ZNF34 2.763
GMEB2 PCSK4 2.755
GMEB2 PPOX 2.74
GMEB2 REEP6 2.726
GMEB2 COG8 2.724
GMEB2 HYAL2 2.72
GMEB2 NIP7 2.718
GMEB2 RPL10 2.678
GMEB2 WDR46 2.674
GMEB2 PFDN6 2.673
GMEB2 UFC1 2.662
GMEB2 ZNF497 2.655
GMEB2 RASSF1 2.653
GMEB2 RPS2 2.651
GMEB2 H2BC6 2.65
GMEB2 RPL26 2.647
GMEB2 VPS11 2.646
GMEB2 ZNF837 2.645
GMEB2 RPS5 2.644
GMEB2 ZNF668 2.641
GMEB2 TP53I3 2.637
GMEB2 ZNF646 2.629
GMEB2 RXRB 2.621
GMEB2 SLC39A7 2.62
GMEB2 HYAL1 2.614
GMEB2 H4C4 2.609
GMEB2 EID2 2.605
GMEB2 YDJC 2.604
GMEB2 PSMB6 2.601
GMEB2 CDC37 2.6
GMEB2 TBCB 2.6
GMEB2 H2AX 2.592
GMEB2 NDUFB10 2.592
GMEB2 PIH1D1 2.592
GMEB2 ZBTB45 2.589
GMEB2 TRMT112 2.579
GMEB2 ZC3H4 2.577
GMEB2 FKBP2 / LOC114841035 2.575
GMEB2 LOC114841035 2.575
GMEB2 FOS 2.575
GMEB2 POLR2I 2.568
GMEB2 ALDH16A1 2.553
GMEB2 NUP155 2.552
GMEB2 IDE 2.548
GMEB2 DCTPP1 2.548
GMEB2 STX5 2.545
GMEB2 MNT 2.545
GMEB2 RSRP1 2.543
GMEB2 BICRA 2.542
GMEB2 GNL1 2.542
GMEB2 SYF2 2.54
METLL1 METTL14 4.893
METLL1 C12orf57 1.67
METLL1 PTPN6 1.387
METLL1 TEX14 1.374
METLL1 LOC730183 1.057
METLL1 FBRS 1.04
METLL1 SRCAP 1.007
METLL1 EIF2B3 1
METLL1 TRMT2A 0.999
METLL1 DNAJC11 0.999
METLL1 ARHGAP24 0.999
METLL1 CDCA5 0.998
METLL1 CLUAP1 0.998
METLL1 C7orf26 0.998
METLL1 NIPSNAP2 0.997
METLL1 ARHGEF2 0.997
METLL1 THEM6 0.997
METLL1 SLC45A4 0.997
METLL1 TAF12 0.996
METLL1 R3HCC1 0.995
METLL1 BAIAP2 0.995
METLL1 DRAP1 0.995
METLL1 POLR2I 0.995
METLL1 ABHD16A 0.995
METLL1 RAB37 0.995
METLL1 DHX16 0.994
METLL1 FOXP4 0.993
METLL1 AURKAIP1 0.993
METLL1 KRBA2 0.993
METLL1 SUPT4H1 0.993
METLL1 PPIL2 0.992
METLL1 KCTD5 0.992
METLL1 ZFPL1 0.992
METLL1 TMEM14B 0.99
METLL1 DPP9 0.99
METLL1 RANBP1 0.99
METLL1 TMEM44 0.989
METLL1 SRI 0.989
METLL1 NOTUM 0.988
METLL1 SLC9A1 0.988
METLL1 PGK1 0.988
METLL1 LEMD1 0.987
METLL1 NDUFS8 0.987
METLL1 WDR74 0.987
METLL1 FAM83D 0.986
METLL1 ZNF341 0.986
METLL1 POLG 0.985
METLL1 SH2B1 0.984
METLL1 C11orf68 0.98
METLL1 LARP1 0.977
METLL1 TTI2 0.975
METLL1 POLDIP3 0.971
METLL1 CCDC200 0.968
METLL1 TBCB 0.964
METLL1 LRP5 0.959
METLL1 PTK6 0.954
METLL1 SIRT4 0.945
METLL1 RAB27A 0.898
METLL1 MTRNR2L1 0.893
METLL1 ZFPM1 0.861
METLL1 UCKL1 0.847
METLL1 SCIMP 0.845
METLL1 KIF2C 0.845
METLL1 FOXJ1 0.835
METLL1 NEU1 0.834
METLL1 ATN1 0.826
METLL1 ZFAND2A 0.816
METLL1 PARP16 0.814
METLL1 TMEM107 0.812
METLL1 SEMA6A 0.795
METLL1 RGS3 0.785
METLL1 OVOL3 0.781
METLL1 LY6G6F 0.778
METLL1 LY6G6F-LY6G6D 0.778
METLL1 LOC107986004 0.772
METLL1 CNOT3 0.76
METLL1 C16orf90 0.742
METLL1 ANKRD39 0.739
METLL1 PPARD 0.731
METLL1 ABCC1 0.724
METLL1 GUCD1 0.721
METLL1 ABCA4 0.71
METLL1 TMEM262 0.707
METLL1 ARID3B 0.703
METLL1 FGG 0.702
METLL1 SNRPD3 0.696
METLL1 ZDHHC18 0.692
METLL1 SEMA4C 0.685
METLL1 PTF1A 0.683
METLL1 CLPTM1L 0.674
METLL1 SOCS2 0.669
METLL1 SREBF1 0.655
METLL1 LMNB2 0.65
METLL1 ANKRD35 0.647
METLL1 PRR14 0.647
METLL1 RPL26 0.643
METLL1 ACSL4 0.62
METLL1 USP48 0.617
METLL1 ECM1 0.591
METLL1 NEU4 0.59
NGF ANTKMT 7.618
NGF METRN 7.608
NGF CCDC78 7.085
NGF LFNG 7.027
NGF EFNA4 6.879
NGF NEMP1 6.673
NGF HAGHL 6.654
NGF ZBTB42 6.594
NGF PAQR4 6.577
NGF EFNA3 6.555
NGF FBXL16 6.553
NGF NAB2 6.514
NGF KISS1R 6.479
NGF R3HDM4 6.36
NGF AKT1 6.24
NGF SYTL1 6.211
NGF SH2B2 6.209
NGF NR1D1 6.18
NGF CALR 6.143
NGF GADD45B 6.138
NGF KREMEN2 6.116
NGF FIS1 6.101
NGF PHYHIP 6.077
NGF PCNX3 6.07
NGF MAP3K11 6.069
NGF ARID3A 6.051
NGF KIAA1522 6.046
NGF PPP1R14B 6.019
NGF CHD4 5.991
NGF FKBP2 5.982
NGF FNDC11 5.965
NGF RAD23A 5.959
NGF ABCB9 5.953
NGF OGFOD2 5.952
NGF LOC114841035 5.948
NGF TAS1R3 5.943
NGF PKMYT1 5.935
NGF TAPBP 5.918
NGF RNF224 5.905
NGF PTH2 5.873
NGF NFIC 5.872
NGF LRRC71 5.855
NGF RILP 5.852
NGF CISH 5.83
NGF SHMT2 5.827
NGF MAPKAPK3 5.827
NGF SCARF1 5.817
NGF CYSRT1 5.815
NGF MAPK12 5.811
NGF GFY 5.81
NGF FARSA 5.766
NGF SLC34A3 5.758
NGF RNF208 5.739
NGF LOC112267874 5.729
NGF ZBTB22 5.727
NGF ADAM15 5.698
NGF TTC9B 5.682
NGF KCNN1 5.669
NGF INTS11 5.661
NGF CPTP 5.66
NGF CLCF1 5.645
NGF SLC38A3 5.645
NGF AGPAT2 5.635
NGF ARL6IP4 5.634
NGF SP2 5.629
NGF OAZ1 5.624
NGF REXO1 5.623
NGF SERTAD1 5.619
NGF NOS3 5.617
NGF SERPINE1 5.611
NGF NENF 5.603
NGF CLDN15 5.599
NGF HOXB4 5.598
NGF WDR24 5.595
NGF VASP 5.592
NGF LBHD1 5.578
NGF UQCC3 5.577
NGF MAPK11 5.575
NGF PDE4A 5.573
NGF SLC37A4 5.558
NGF GFER 5.535
NGF NKX2-2 5.533
NGF MAPK7 5.531
NGF EHD2 5.524
NGF NOXO1 5.521
NGF B9D1 5.515
NGF SRMS 5.501
NGF PLCB3 5.497
NGF GADD45GIP1 5.492
NGF ARHGAP45 5.464
NGF UCKL1 5.455
NGF KCNK7 5.447
NGF PLEC 5.439
NGF PRKAR1B 5.437
NGF SLC12A5 5.426
NGF ELFN1 5.416
NGF CLDN9 5.41
NGF SPINT2 5.409
NGF SECTM1 5.409
NGF NEUROG2 5.401
NGF CHTF18 3.653
NGF TRIM39 3.306
NGF KRT17 1.777
NGF PGF 1.479
NGF FOXJ1 1.431
NGF KNTC1 1.3
NGF F2R 1.202
NGF POLR3G 1.012
POLR3G HMGN4 4.191
POLR3G ALOXE3 3.96
POLR3G HES7 3.844
POLR3G TRIM7 3.068
POLR3G SHF 2.792
POLR3G TRIM41 2.5
POLR3G ANG 2.233
POLR3G RNASE4 2.175
POLR3G BORCS6 1.997
POLR3G PGBD2 1.959
POLR3G ZNF184 1.944
POLR3G AGBL5 1.941
POLR3G TRIM55 1.933
POLR3G HSPA6 1.93
POLR3G DPP9 1.926
POLR3G POLR3E 1.908
POLR3G LOC105375107 1.759
POLR3G POM121L2 1.755
POLR3G PLXDC1 1.678
POLR3G ABT1 1.652
POLR3G OR6S1 1.606
POLR3G UBC 1.598
POLR3G H4C8 1.553
POLR3G AURKB 1.529
POLR3G PER1 1.471
POLR3G LASP1 1.429
POLR3G TRIM27 1.36
POLR3G HJV 1.349
POLR3G TMEM107 1.284
POLR3G PRSS16 1.28
POLR3G MPZL1 1.245
POLR3G H2BC17 1.05
POLR3G MDM4 1.032
POLR3G H2AC17 1.032
POLR3G OR2B2 1.026
POLR3G VAMP2 1.025
POLR3G CGB2 0.994
POLR3G KNL1 0.991
POLR3G TIA1 0.986
POLR3G ILF2 0.983
POLR3G PLEKHG2 0.982
POLR3G FBXO31 0.98
POLR3G DCT 0.98
POLR3G POLG 0.979
POLR3G SACM1L 0.978
POLR3G PRMT5 0.978
POLR3G CCDC107 0.977
POLR3G LUC7L2 0.975
POLR3G RAB11A 0.974
POLR3G BCAR3 0.974
POLR3G METTL26 0.973
POLR3G FMC1-LUC7L2 0.972
POLR3G NDUFS7 0.972
POLR3G LTA4H 0.97
POLR3G TMEM259 0.969
POLR3G SLC27A4 0.968
POLR3G C8orf37 0.966
POLR3G B4GAT1 0.965
POLR3G PPP6R3 0.962
POLR3G FMC1 0.959
POLR3G FCGR3A 0.958
POLR3G NCOA7 0.956
POLR3G RPL23 0.955
POLR3G SMARCC2 0.941
POLR3G PPEF1 0.936
POLR3G MYNN 0.921
POLR3G ZMAT2 0.916
POLR3G H2BC13 0.905
POLR3G ZNF213 0.903
POLR3G TMPO 0.891
POLR3G MTRNR2L1 0.888
POLR3G H2AC13 0.888
POLR3G H3C12 0.875
POLR3G ARL5C 0.872
POLR3G PIK3C2B 0.87
POLR3G ACTRT3 0.832
POLR3G RNASE12 0.827
POLR3G RS1 0.821
POLR3G BRMS1 0.811
POLR3G KRBA1 0.808
POLR3G FCGR3B 0.806
POLR3G RNASE11 0.795
POLR3G TMEM214 0.789
POLR3G H3C10 0.781
POLR3G LOC101929805 0.742
POLR3G ZNF692 0.722
POLR3G C1QTNF6 0.719
POLR3G PSMA3 0.713
POLR3G CCDC115 0.709
POLR3G CNN2 0.707
POLR3G IMP4 0.705
POLR3G MCRIP2 0.704
POLR3G OR1F1 0.7
POLR3G CGB1 0.7
POLR3G ZFP36 0.685
POLR3G PLLP 0.678
POLR3G PFAS 0.674
POLR3G WFIKKN1 0.67
POLR3G CTC1 0.653
POLR3G H2BC10 0.652
SVIL H4C2 1.915
SVIL H3C6 1.817
SVIL H2BC8 1.813
SVIL H3C1 1.805
SVIL H2AC8 1.804
SVIL H4C1 1.795
SVIL PGBD3 1.77
SVIL H2AC12 1.58
SVIL H4C9 1.58
SVIL H2BC12 1.574
SVIL H3C2 1.569
SVIL H1-1 1.504
SVIL H2AC4 1.498
SVIL H3C3 1.482
SVIL H2BC3 1.427
SVIL H4C4 1.375
SVIL H2AC6 1.356
SVIL H2BC9 1.355
SVIL CROCC 1.355
SVIL H2BC4 1.342
SVIL H3C7 1.298
SVIL H4C3 1.288
SVIL TMEM135 1.263
SVIL H2BC5 1.259
SVIL LUC7L2 1.25
SVIL FMC1-LUC7L2 1.244
SVIL FMC1 1.234
SVIL H1-3 1.178
SVIL GEMIN6 1.155
SVIL H4C7 1.153
SVIL SRSF7 1.153
SVIL H1-4 1.142
SVIL H1-6 1.137
SVIL TEX14 1.135
SVIL MAK16 1.131
SVIL H4C8 1.12
SVIL TTI2 1.119
SVIL H4C5 1.104
SVIL TNFRSF10D 1.088
SVIL H4C6 1.073
SVIL TIMM13 1.06
SVIL H2BC6 1.054
SVIL H3C4 1.053
SVIL H2AC7 1.053
SVIL H2BC7 1.052
SVIL CSDE1 1.052
SVIL NRAS 1.044
SVIL TET1 1.031
SVIL CUEDC2 1.031
SVIL H2AC11 1.027
SVIL ZSCAN12 1.025
SVIL ALOXE3 1.022
SVIL FRG1 1.012
SVIL RPS27L 1.011
SVIL H2BC11 1.01
SVIL PSMB6 1.009
SVIL COA6 1.009
SVIL EIF3E 1.008
SVIL DSTYK 1.007
SVIL MRPS35 1.005
SVIL MCOLN1 1.004
SVIL COX7A2 1.003
SVIL FBXW9 1.001
SVIL IST1 1
SVIL ACCS 1
SVIL HSP90AB1 1
SVIL ANAPC15 1
SVIL UBB 1
SVIL LRRC63 1
SVIL JMJD1C 1
SVIL PHB 1
SVIL EPG5 1
SVIL FUS 1
SVIL PSMC2 1
SVIL SYNPO2 1
SVIL MNAT1 0.999
SVIL GOSR1 0.999
SVIL ITPR1 0.999
SVIL TDG 0.999
SVIL FBXW7 0.999
SVIL EEF1A1 0.999
SVIL MTERF1 0.999
SVIL SF3A3 0.999
SVIL RABGAP1L 0.999
SVIL NDUFAF1 0.999
SVIL RPL12 0.999
SVIL NSL1 0.999
SVIL JUNB 0.999
SVIL COX14 0.999
SVIL LRRC37A3 0.999
SVIL EMC4 0.999
SVIL HNRNPA2B1 0.999
SVIL RPL7 0.999
SVIL RPS27 0.999
SVIL RPL10 0.998
SVIL HSPA1A 0.998
SVIL DVL2 0.998
SVIL RPS6 0.998
SVIL MAGED1 0.998
SVIL ZNHIT6 0.998
TOE1 LOC101929601 3.499
TOE1 LOC101929627 3.235
TOE1 ZDHHC7 2.861
TOE1 RASA4 2.803
TOE1 ARID1A 2.729
TOE1 PPP2R5B 2.595
TOE1 ATG2A 2.582
TOE1 MNT 2.545
TOE1 MRPL48 2.493
TOE1 MCRIP2 2.47
TOE1 H2AX 2.433
TOE1 TSEN34 2.422
TOE1 MBOAT7 2.407
TOE1 MFAP3L 2.379
TOE1 MRPL44 2.347
TOE1 WDR90 2.338
TOE1 MRPL40 2.333
TOE1 HIRA 2.314
TOE1 DPAGT1 2.295
TOE1 EIF2B5 2.221
TOE1 METTL26 2.206
TOE1 FGF9 2.193
TOE1 RGL2 2.168
TOE1 PYGO2 2.164
TOE1 HMBS 2.156
TOE1 LOC101928120 2.155
TOE1 PBXIP1 2.141
TOE1 RTEL1 2.131
TOE1 SLC25A39 2.103
TOE1 VILL 2.095
TOE1 NEU1 2.091
TOE1 TRIP6 2.088
TOE1 LOC107985728 2.088
TOE1 AMDHD2 2.084
TOE1 FZR1 2.072
TOE1 KIAA0513 2.029
TOE1 ACAP2 2.022
TOE1 SYTL1 2.017
TOE1 FUT1 2.014
TOE1 KIAA0586 2.01
TOE1 TIMM9 2.01
TOE1 GOLGA6L10 2.01
TOE1 CAMK1 1.993
TOE1 SAMD11 1.992
TOE1 UBC 1.986
TOE1 NOTCH2NLA 1.977
TOE1 RAB7A 1.973
TOE1 C4orf47 1.97
TOE1 UFSP2 1.97
TOE1 AP3S1 1.961
TOE1 MCEE 1.961
TOE1 TMEM250 1.959
TOE1 LRRC1 1.958
TOE1 P3H4 1.958
TOE1 KCNAB2 1.956
TOE1 NPHP4 1.956
TOE1 RBPJ 1.955
TOE1 MPHOSPH10 1.952
TOE1 RAP2B 1.951
TOE1 DNAAF3 1.95
TOE1 MRPL36 1.948
TOE1 DOHH 1.947
TOE1 ERAP1 1.947
TOE1 RPS9 1.943
TOE1 SEPTIN2 1.941
TOE1 CITED2 1.941
TOE1 HDLBP 1.94
TOE1 ATG12 1.94
TOE1 ZFAND6 1.937
TOE1 TDP1 1.936
TOE1 LNPEP 1.935
TOE1 FAM72D 1.931
TOE1 ZFP91 1.926
TOE1 ATP6V0C 1.922
TOE1 EEF2 1.922
TOE1 TRIM65 1.919
TOE1 CCNT2 1.918
TOE1 C2 1.918
TOE1 EHMT2 1.918
TOE1 EFCAB11 1.916
TOE1 GANAB 1.916
TOE1 ISOC2 1.914
TOE1 ANP32B 1.914
TOE1 SLC12A9 1.913
TOE1 COMMD1 1.913
TOE1 NACC1 1.912
TOE1 YPEL5 1.911
TOE1 TRMT1 1.91
TOE1 FKBP10 1.906
TOE1 HSPG2 1.905
TOE1 INTS5 1.902
TOE1 ZMIZ1 1.901
TOE1 ARL5A 1.9
TOE1 PLEKHA8 1.897
TOE1 COIL 1.896
TOE1 MEIS1 1.894
TOE1 SRRT 1.892
TOE1 GATD3B 1.89
TOE1 FKBP14 1.885
TOE1 SRGAP2B 1.883
TRIM3 Pde2a 4.206
TRIM3 Ptp4a3 4.194
TRIM3 Erg 4.052
TRIM3 Cxxc5 3.926
TRIM3 Rassf2 3.738
TRIM3 Chrna9 3.689
TRIM3 LOC115488942 3.652
TRIM3 Cbfa2t3 3.558
TRIM3 Cep250 3.508
TRIM3 Dnah7a 3.483
TRIM3 Cerk 3.431
TRIM3 Rac2 3.411
TRIM3 Plxnd1 3.384
TRIM3 Zbtb18 3.369
TRIM3 Laptm5 3.32
TRIM3 Adssl1 3.316
TRIM3 Pomgnt2 3.221
TRIM3 N4bp3 3.216
TRIM3 Smad3 3.206
TRIM3 Rmnd5b 3.195
TRIM3 Iqcn 3.182
TRIM3 Cd52 3.168
TRIM3 Cyth4 3.162
TRIM3 Jund 3.161
TRIM3 Unc93b1 3.117
TRIM3 Tfap4 3.098
TRIM3 Sfi1 3.093
TRIM3 Ttpal 3.091
TRIM3 Tpcn1 3.09
TRIM3 Eef2k 3.078
TRIM3 Aldh3b1 3.075
TRIM3 Gm4832 3.061
TRIM3 Adgre5 2.984
TRIM3 Il2ra 2.932
TRIM3 Fmnl1 2.896
TRIM3 Tfeb 2.886
TRIM3 Wdfy4 2.886
TRIM3 Slamf6 2.88
TRIM3 Gm32211 2.862
TRIM3 Ddit4 2.849
TRIM3 Abca6 2.843
TRIM3 Aff1 2.837
TRIM3 Tpst2 2.816
TRIM3 Ky 2.815
TRIM3 Lrmp 2.787
TRIM3 Srl 2.77
TRIM3 Irgc1 2.761
TRIM3 Ralgds 2.76
TRIM3 AI467606 2.756
TRIM3 Ubxn11 2.733
TRIM3 Pacsin1 2.732
TRIM3 Snn 2.707
TRIM3 Ackr2 2.699
TRIM3 Ppp1r18 2.686
TRIM3 Msh6 2.664
TRIM3 Akna 2.662
TRIM3 Rgs8 2.657
TRIM3 Hdac7 2.647
TRIM3 Emid1 2.628
TRIM3 Fam129c 2.613
TRIM3 Fgd2 2.599
TRIM3 4930467E23Rik 2.598
TRIM3 Gna15 2.598
TRIM3 Rilpl2 2.572
TRIM3 Inf2 2.569
TRIM3 Pxdn 2.567
TRIM3 Scgb1a1 2.549
TRIM3 Gm19402 2.546
TRIM3 Amz2 2.529
TRIM3 H4c14 2.525
TRIM3 Cyth1 2.518
TRIM3 Nr4a1 2.509
TRIM3 Kcnj12 2.496
TRIM3 Cxcr4 2.495
TRIM3 Pxn 2.491
TRIM3 Dgkd 2.489
TRIM3 Enpp6 2.479
TRIM3 Cd79b 2.475
TRIM3 Sike1 2.471
TRIM3 Cdh17 2.434
TRIM3 Nmrk1 2.427
TRIM3 Rbm17 2.421
TRIM3 Arap1 2.42
TRIM3 Ostf1 2.414
TRIM3 Tspear 2.414
TRIM3 P2rx1 2.398
TRIM3 Rnase10 2.394
TRIM3 BC034090 2.387
TRIM3 Tex14 2.381
TRIM3 Ech1 2.378
TRIM3 Bex6 2.378
TRIM3 Asb2 2.373
TRIM3 Rbm38 2.373
TRIM3 Socs3 2.37
TRIM3 Cnn1 2.363
TRIM3 Pgls 2.362
TRIM3 Kcnab2 2.361
TRIM3 Stambpl1 2.357
TRIM3 Lrrc32 2.348
TRIM3 Nrm 2.344

Fig. 5.

Fig. 5

MSCs induce NGF demethylation to regulate the expression of KRT17 and FOXJ1. A MSCs reverse the methylation of transcription factors and the regulatory network of target genes during TEC aging (orange triangles represent transcription factors, and blue circles represent target genes). B The transcription factor NGF was associated with the RNA-seq expression of the target genes KRT17 and FOXJ1 during the reversal of TEC senescence by MSCs. The horizontal coordinate represents the expression of the transcription factor NGF, and the ordinate represents the expression of the target gene. C The mRNA expression levels of demethylated NGF, KRT17 and FOXJ1 were analyzed via qPCR. D The expression of NGF was detected by immunofluorescence (400×). E The expression of the TEC proliferation marker Ki67 was detected by immunofluorescence (400×). F Regulatory role of decreased methylation of the NGF/KRT17/FOXJ1 axis in the MSC-mediated reversal of TEC senescence. The measured values represent the mean ± standard deviation (`X ± s) of three replicate experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

Immunofluorescence analysis and RT‒qPCR further proved that NGF interacted with KRT17 and FOXJ1 (Fig. 5C and D). Functionally, NGF, KRT17 and FOXJ1 were mainly involved in the proliferation of epithelial cells. KI67 showed that the proliferation ability of ageing TECs increased after coculture with MSCs (Fig. 5E). In summary, these results suggest that MSCs demethylate the NGF gene of ageing TECs and promote the expression of KRT17 and FOXJ1, to promote the growth and proliferation of ageing TECs (Fig. 5F).

3. Materials and methods

3.1. Animal

Six macaque monkeys were purchased from Kunming Animal Laboratory Center, Chinese Academy of Sciences (Experimental Animal Production License No.: SCXK (Yunnan) K2017-0003), aged 22–28 years old, Weight 3.5–6.5 kg, normally raised in the Laboratory Animal Center of 920th Hospital of the Chinese People's Liberation Army Joint Logistics Support Force (Laboratory animal license No.: SYXK (Military) 2017-0051) Laboratory animal studies were approved by the Theoretical Committee of the 920th Hospital of the Chinese People's Liberation Army Joint Logistics Support Force, the approval number: 2020-034 (Section) −01.

3.2. MSCs

MSCs of macaque monkeys (BMMSCs) and human (UCMSCs) were derived from the stem cell Bank of the National and Local Joint Engineering Center for Stem Cell and Immune Cell Biomedical Technology affiliated with the 920th Hospital of the Joint Logistic Support Force. The BMMSCs and UCMSCs preserved in liquid nitrogen were rapidly thawed in warm water at 37 °C, normal saline was added, and the mixture was centrifuged at 2000 rpm for 3 min. The cells were resuspended in the DMEM/F12 culture medium containing 10% fetal bovine serum and inoculated in a culture bottle at a density of 5000/cm2, after which the DMEM/F12 culture medium was changed the next day. When the degree of cell fusion reached 85%, the cells were digested with trypsin containing 0.25% EDTA and passaged with DMEM/F12 culture medium. P4 generation BMMSCs and UCMSCs were used to determine their differentiation potential and surface markers, and the detailed methods used were described in our previous published article [19].

3.3. Cell injection

When the fusion degree of the cultured P4 MSCs generation was approximately 80%, the cells were digested and washed. They were diluted with 0.9% sterile sodium chloride solution at a concentration of 2 × 106 cells/ml. Macaque monkeys were randomly divided into two groups: the senescence model group (n = 3) and the senescence treatment group (n = 3), After the macaque monkeys in the treatment group were immobilized, MSCs were injected into the femoral vein at 1 × 107/kg per monkey once every other day 3 times, the senescence model group was infused with isovolumetric saline at the same time.

3.4. Thymus index measurement

Five months after the treatment, macaques in each group were fasted for 24 h. The body weight of the macaques in each group was measured (kg), and the monkeys were anesthetized with excess 3% sodium pentobarbital (1 kg/ml). The abdominal cavity was exposed, the rib cartilage on both sides was cut up to the upper margin of the thoracic cavity, and the thymus tissue was exposed below the heart. An electronic balance was used to weigh the mass (g) and calculate the thymus index of each monkey individually and the average value of each group was taken for statistical analysis. The calculation formula is as follows:

Thymusindex=Thymusmassx(g)Macaquebodyweight(kg)×100%

3.5. Thymus tissue HE staining

The thymus tissue was fixed in 4% paraformaldehyde solution for 24 h and then dehydrated and embedded in paraffin. The thickness of the sections was approximately 4 μm. After being treated with xylene-anhydrous ethanol-75% ethanol, the sections were sealed with neutral glue. Sections were observed and photographed with a Nikon microscope (NikonDS-U3, Nikon, Japan).

3.6. Determination of thymosin α and thymopoietin levels

Five months after cell injection, 5 ml of blood was collected from the femoral vein into a heparin tube and centrifuged at 2000 rpm for 10 min, after which the obtained serum was transferred to a microcentrifuge tube. The expression levels of thymosin α and thymopoietin in the serum were measured by ELISA kits (Millipore). The procedure was performed according to the reagent instructions. The absorbance (OD) of each well was measured at a wavelength of 450 nm in sequence. The measurement was carried out within 15 min after adding the termination solution. With the concentration of the standard product as the horizontal coordinate and the OD value as the vertical coordinate, a standard curve was drawn, and the corresponding concentration was determined according to the OD value of the sample multiplied by the dilution factor, which was the actual concentration of the sample.

3.7. Detection of P16 expression by immunofluorescence staining

The thymus tissue was fixed in 4% paraformaldehyde solution for 24 h and then dehydrated and embedded in paraffin. The thickness of the section was approximately 4 μm. Primary antibody Rabbit anti-monkey antibody P16 (Serviceebio:GB111143) was added for staining. FITC goat anti-rabbit was incubated at room temperature for 50 min, and the nuclei were restained with DAPI for 15 min. Finally, the sections were sealed with anti-fluorescence quenching sealant and photographed under a microscope (Nikon DS-U3, Nikon, Japan).

3.8. Immunohistochemical staining of the thymic epithelial cell-specific marker molecule CK14

Thymus tissue sections with a thickness of 4 μm were dewaxed in water for antigen repair and blocking of endogenous peroxidase. Primary antibody Mouse anti-monkey antibody CK14 antibody (Servicebio: GB12803-100) was used, and the second antibody was labeled with HRP. After incubation with the secondary antibody, the nuclei were stained with DAPI (Servicebio: G1012; DAPI dyeing: The slides were placed in PBS (pH 7.4) and washed by shaking on a decolorizing shaker 3 times for 5 min each. DAPI dye was added, and the samples were incubated at room temperature for 10 min in the dark. Sealing: The slide was placed in PBS (pH 7.4) and washed by shaking on the decolorizing 3 times for 5 min each. Anti-fluorescence quenching sealing tablets were used.). Sections were observed and photographed with a Nikon microscope (Nikon DS-U3, Nikon, Japan). The positive area signal was analyzed by ImageJ software (NIH, Bethesda, MD).

3.9. TECs

TEC (species: human) is a standard cell line obtained from Meisen CTCC, Catalogue No.: CTCC-004-0022p2. After the acquisition, DMEM/F12 medium containing 10%FBS was used in our laboratory for passage culture. The growth morphology of the cells was cobblestone. Meanwhile, EPCAM, a specific marker of high expression on the surface, was identified by immunofluorescence, which was consistent with the standard TEC reported in the literature, and the accuracy of the cells was confirmed (Supplementary Fig.1).

3.10. Establishment and identification of the TEC ageing model and a coculture model of MSCs

Fourth-generation TECs were collected, the cell concentration was adjusted to 5 × 105 L-1, and 100 μL of cell suspension was inoculated in a 6-well culture plate. When the cell density was approximately 80%, 200 μmol/L H2O2 (China Nanguo Pharmaceutical Co., Ltd.) was added, and the cells were cocultured for 72 h. Fourth-generation MSCs were collected, the cell concentration was adjusted to 5 × 105 L-1, 100 μL of cell suspension was inoculated into the Transwell chamber, the Transwell chamber was embedded in the upper layer of the 6-well culture plate, and DMEM/F12 containing 10% fetal bovine serum was used for coculture of cells for 48 h.

3.11. SA-β-gal (senescence-associated β-galactosidase) staining

TECs were collected, washed with PBS once, operated according to the kit instructions (Solarbio, G1580), and observed under an ordinary optical microscope (ZEN2011), and the number of blue-positive cells in three visual fields was counted.

3.12. Expression level analysis of P21 and P16

TECs was collected for Western blotting analysis and lysed in RIPA buffer (Solarbio, China, Beijing). The total proteins of TEC cells were separated by Tris-Gly and 10% gel electrophoresis and then transferred to a nitrocellulose membrane. The cell membrane was blocked with 5% skim milk at room temperature for 1.5 h and then incubated with an antibody overnight. The next day, the membrane was washed with PBST and incubated for 30 min with horseradish peroxidase-labeled secondary antibody. Protein bands were detected after treatment with SuperSignal West Femto (Thermo Fisher Science, Waltham, MA, USA). Primary anti-rabbit anti-human P16 (1RV 1000, ab32503), P21 (1RV 1000, ab32351), SRC (1RV 300000 ab179473 ab109381), and ITGB3 (1RV 1000 ab179473) antibodies were obtained from Abcam. The primary anti-rabbit anti-human β-actin antibody (1RV 25000∼66009-1-Ig) and the secondary anti-HRP-Goat IgG antibody (1RV 1000, SA00001-2) were purchased from Proteintech.

3.13. Cell apoptosis assay

TUNEL staining was used to determine the apoptosis of TECs. TECs were collected, paraformaldehyde was fixed, the fixating solution was removed, 100 μl film breaking working solution was added, incubated at room temperature for 20 min, washed 3 times with PBS for 5 min each time, and buffer was added to cover the tissue for 10 min. Appropriate amounts of TDT enzyme and dUTP in the TUNEL kit were mixed with buffer at a 1:5:50 ratio. The slides were incubated for 2 h in an incubator at 37 °C, DAPI dye solution was added, the slides were incubated for 10 min at room temperature in the dark, and the slides were sealed with anti-fluorescence quenching tablet sealing agent. Microscopic photography: Sections were observed under a fluorescence microscope, and images were collected. DAPI emits blue light at 420 nm, while CY3 emits red light at 590 nm.

3.14. Observation of the cell substructure by fluoroscopic electron microscopy

TECs were collected, digested with 0.25% trypsin and made into a cell suspension. After washing with PBS, the cell precipitate was centrifuged and fixed for 4 h. After immersion in acetone, ultrathin sections of 80 nm were stained with uranium and lead, observed under a transmission electron microscope (Hitachi, HT7700), and photographed for image analysis.

3.15. Continuous observation of the morphology of cocultured TECs by a Nanolive 4D microscope

P4 generation TECs at a cell concentration of 5000/ml were added to a Nanolive 4D microscope petri dish, and then 1.5 ml DMEM/F12 (containing 10% fetal bovine serum) medium was added and mixed slowly; the medium was discarded after 24 h of culture. Then, DMEM/F12 containing 200 μmol/L H2O2 was added to treat the TECs for 72 h, after which the medium was discarded. Next MSCs at a P4 generation cell concentration of 5000 cells/ml were added and incubated for 48 h The Nanolive 4D microscope (Nikon Eclipse C1) was started and connected to the computer, the focal length was adjusted to take pictures under the appropriate field of vision for 48 h, and pictures were taken every 4 min.

3.16. DNA methylation and transcriptome sequencing

The TECs of MSCs were 1.3 × 107 in coculture and 1.2 × 107 in the induced senescence group. The cell precipitates were frozen in liquid nitrogen and stored at −80 °C for 24 h. Whole-genome methylation and transcriptome data were obtained by whole-genome methylation and transcriptome sequencing at Guangzhou Jidiao Biotechnology Co., Ltd (2009).

3.17. Analysis of DNA methylation sequencing data

The obtained clean data were mapped to the species reference genome using BSMAP software (version 2.90). The methylated cytosine level was then detected using a custom Perl script and assessed using the correction algorithm described by Lister R et al. The level of methylation was calculated based on the percentage of methylation of each sequence (CG, CHG, and CHH) in the entire genome, each chromosome, and different regions of the genome. To explain the changes in DNA methylation in TECs, methylation maps of the upstream 1 kb region and gene body were drawn according to the average methylation level of each window. Changes in GC content were screened according to the number of differentially methylated regions (DMRs) (≥5) and the absolute value of methylation ratio difference (≥0.1). The senescence genes were counted according to Gene Ontology Consortium.

3.18. RNA-seq analysis

The transcriptome database was analyzed by DESeq2 software, and the mRNAs satisfying the conditions of log2FC > 1.2 and P value < 0.05 were regarded as differentially expressed genes. The differentially expressed genes in ageing TECs and ageing TECs after coculture were counted and analyzed by gene function annotation.

3.19. Analysis of the relationship between gene methylation and transcriptional level

To calculate the correlation between gene methylation and transcription, the genes were divided into four groups according to the transcription level in RNA-seq data (FPKM ≤1 for no transcription, 1 < FPKM ≤10 for low transcription, 10 < FPKM ≤100 for medium transcription and FPKM >100 for high transcription). The Pearson correlation coefficients between upstream/gene body methylation levels and corresponding gene expression levels in TECs of the control, model and coculture groups were calculated.

3.20. Establishment of the transcription factor-methylation regulatory network

To explore the relationship between specific gene methylation and transcription factor binding, the TFs of ageing-related genes were predicted by CistromeDB, and the TF-DNA regulatory network was constructed by Cytoscape.

3.21. Enrichment analysis

Through the Gene Ontology database (GO item in http://www.geneontology.org/)), the number of genes in each item was calculated, and the GO items significantly enriched relative to the genomic background were defined by hypergeometric test. KEGG is the main public pathway-related database (http://www.kegg.jp/kegg/)).

3.22. Functional verification

3.22.1. Expression analysis of RAP1 signal pathway molecules RAP1, SRC and ITGB3

For Western blotting analysis, the primary anti-rabbit anti-human RAP1 (1RV 1000, ab14404), SRC (1RV 30000 ab179473 ab109381) and ITGB3 (1RV 1000 ab179473) were all obtained from Abcam, as described above.

3.22.2. Analysis of the relative expression of the ageing regulatory genes NGF, KRT17 and FOXJ1

Total RNA was extracted according to the production manual (Carlsbad Invitrogen Company, California) by RT-QPCR using TRIzol reagent. cDNA was amplified by reverse transcriptase (GoldenstarTM RT6, China) and random primers. According to the SYBR Green Master Mix specification (TsingKe Biotech Co., China), a CFX96TM Real-Time System (Applied 2720) was used for qPCR. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Each experiment was repeated 3 times, and the gene expression level was normalized by the 2-T square method. All qPCR primers are listed in (Supplementary Table 1).

3.22.3. For immunofluorescence detection of NGF and KI67 expression

TECs were collected and fixed with paraformaldehyde, and the fixation solution was removed. Anti-mouse anti-human antibodies against Ki67 and NGF (1R1000 ∼ (th) ServiceBio) were added for staining. The nuclei at 15 min were restrained with CY3 goat anti-rabbit DAPI at room temperature for 50 min and then sealed with anti-fluorescence quenching sealant. Microscopic examination: The sections were observed under a fluorescence microscope, and images were collected (Nikon DS-U3, Nikon, Japan; DAPI UV excitation wavelength 380 nm, emission wavelength 420 nm, blue light; CY3 excitation wavelength 510–560 nm, emission wavelength 590 nm, red light). The number of positive cells was analyzed by ImageJ software (NIH, Bethesda, MD).

3.23. Statistical analysis

SPSS 21.0 statistical software was used for analysis, and the data are expressed as the mean ± standard deviation. A t-test was used to compare the two groups, and one-way analysis of variance (one-way ANOVA) was used to analyze the significance of differences among the three groups.

4. Discussion

Thymus aging weakens the immune system and other functions of the human body and is a progressive, multifactorial and complex biological process caused by serious damage to the microenvironment and integrity of the thymus matrix. MSC transplantation, as a new method for the treatment of thymus aging, has been proven to be effective and safe, and it has been proposed as a treatment to restore the structure and function of the thymus; for example, MSCs significantly increase the thymus index and improve the histological structure of the aging thymus [14]. Similarly, the structure was reconstructed, and the function of the aging thymus was improved after continuous MSC infusion [20]. However, in-depth research on this process in primates is lacking. Interestingly, we found that MSCs could regulate DNA methylation profiles to promote the division and proliferation of aging TECs, thereby improving the structure and function of the thymus in aging macaques.

Rhesus monkey belongs to non-human primates, whose genome sequencing is 93% similar to human genome, and the research results are more valuable for reference than other animals. In this study, we used elderly rhesus monkeys as a model to observe the effect of MSCs on thymus structure and function. To our surprise, our results showed that the volume of the thymus increased, the number of thymus lobules increased, the number of thymus epithelial cells and thymocytes increased, and the number of Hassall bodies and the amount of adipose tissue decreased. Moreover, the thymus index and the levels of thymosin α and thymopoietin in peripheral blood were decreased, the aging marker gene P16 was downregulated, and the TEC marker molecule CK14+ was upregulated after MSC treatment. These results implied that MSC treatment improved the structure and function of the thymus and promoted the regeneration of the thymic lobules.

The current consensus is that the therapeutic mechanism of MSCs involves its homing and strong paracrine functions [21,22]. Our results were consistent with previous studies showing that MSCs promote thymus regeneration by secreting various cytokines or differentiating into thymus tissue cells [23,24]. The above results suggest that MSC treatment improves the structure and function of the thymus in elderly rhesus monkeys.

As TECs are among the most important supporting cells of the thymus, their activity and quantity are crucial to the structural maintenance and function of this organ [25]. Therefore, we used human TECs as the research object, induced aging by H2O2, and observed the effect of intervention with MSCs. H2O2 is a small molecule that induce cell senescence due to oxidative stress caused by high levels of free radicals produced by the cell membrane, and increase the activity of SA-β-gal and expression of P16 and P21 [[26], [27], [28]], which were consisted with our results that the changes of TECs after treatment with 200 μmol/L H2O2 for 72 h. Interestingly, when senescent TECs were cocultured with MSCs in Transwell chambers for 48 h, the activity of SA-β-gal decreased, and P16 and P21 down-regulated, chromatin was evenly distributed, and autophagosomes were observed. Moreover, under a Nanolive 4D microscope, it was found that MSCs continuously secreted vesicular substances into ageing TECs, resulting in uniform distribution of TEC chromatin, appearance of intracellular autophagosomes and smaller cell morphology. These results suggested that MSCs would secrete vesicular substances improve the structure, and down-regulate ageing relative markers to reverse the ageing TECs.

DNA methylation plays an important role in regulating cell structure and function, but whether MSCs regulate DNA methylation to promote the division and proliferation of TECs has not been studied in depth [29,30]. Based on the analysis of the results of genome-wide methylation sequencing, it was found that the sites of methylation modification were mainly concentrated in CG loci, which was consistent with reports in mammals [31]. Then, we analyzed the methylation level of different positions of the gene and observed significant hypomethylation around the transcriptional start site, results showed that the methylation level of the gene body was higher than that of the adjacent region, and the methylation level decreased significantly after the transcriptional end site, indicating that the methylation level changed differently at different locations of the gene [32,33]. In addition, The results of differential methylation gene analysis between groups showed that 23 genes that inhibited cell growth and proliferation were methylated, and 37 genes that promoted cell growth and anti-inflammatory and antioxidant stress were demethylated in TECs after MSCs coculture, which indicated that methylation in the promoter region is one of the key mechanisms by which MSCs reverse TEC senescence.

It has been confirmed that DNA methylation in the gene promoter region can lead to gene silencing [32]. Our study revealed that the methylation of the ageing-promoting genes PDE5A, DUOX2, LAMP1 and SVIL in the promoter region leads to a decrease in the transcription level, while a decrease in the methylation level of the growth-promoting genes POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 leads to an increase in the transcription level. The Rap1 signaling pathway plays an important role in cell adhesion and integrin function in many cell types [34]. After coculture with MSCs, it activates the Rap1 signaling pathway, increases the expression of Rap1, SRC and ITGB3, promotes cell proliferation and facilitates resistance to apoptosis [[35], [36], [37]]. In the gene body region, although some studies have confirmed the effect of gene body DNA methylation on gene expression, the function of gene body DNA methylation is still poorly understood [38,39]. In our study, MSCs in the gene body region not only inhibited gene transcription but also promoted the transcription of some genes in the process of reversing TEC ageing.

DNA methylation in the promoter region prevents binding to transcription factors, leading to gene silencing [40]. Our research shows that NGF gene methylation inhibits the expression of KRT17 and FOXJ1, which highlights the regulatory mechanism of DNA methylation in the process of MSCs coculture with senescent TECs. Nerve growth factor NGF is reported to exist mainly in medullary epithelial cells of normal and regenerated thymic Hassall bodies, stimulating the activity and proliferation of mouse TECs in vitro [41,42]. Therefore, NGF may be a candidate regulatory gene in the process of MSCs coculture of ageing TECs. In addition, KRT17 and FOXJ1, as downstream regulators of NGF, are involved in epithelial cell development, but no studies describing the effects of these genes on TEC senescence have been published, so they may be new markers of TEC cell adhesion, cell proliferation and tissue development.

5. Conclusion

MSCtreatment significantly improved the morphology and structure of the thymus, reduced the expression level of aging markers, and promoted the activity of aging TECs to improve thymus structure and function. The mechanism by which MSCs promote thymus regeneration involves inhibition of the expression of senescence genes by inducing methylation in the promoter region of aging-related genes and inhibiting promoter methylation in cell growth- and differentiation-related genes to regulate their expression. Furthermore, MSCs upregulate KRT17 and FOXJ1 by increasing the expression of genes related to the RAP1 signaling pathway and regulate the demethylation of NGF genes to promote the proliferation of TECs.

Ethics approval and consent to participate

All experimental protocols were approved by the relevant committees of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The datasets [GENERATED/ANALYZED] for this study can be found in the [https://www.jianguoyun.com/p/DTP_bNkQgbjoCxj36ZMFIAA] and GSA -gsa-human (HRA005210).

Funding

This work was supported by two major Science and Technology Special Projects of Yunnan Science and Technology Program (2018ZF001) and (202002AA100006), and Yunnan Province basic research project (202301AT070302). The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Authors’ contributions

XHP and XJW designed the study. ZLY, TC, XQZ, HJ, PH, LY, GPR performed the experiments and collected the data. ZLY wrote the manuscript. TC and ZXH assisted with the literature searches and revised the manuscript. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank everyone on our team for assisting with the preparation of this manuscript.

Footnotes

Peer review under responsibility of the Japanese Society for Regenerative Medicine.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2024.03.008.

Contributor Information

XiJun Wu, Email: 498682999@qq.com.

Xinghua Pan, Email: xinghuapankm@163.com.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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Multimedia component 2
mmc2.xlsx (8.8KB, xlsx)

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Supplementary Materials

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Data Availability Statement

All data generated or analyzed during this study are included in this published article. The datasets [GENERATED/ANALYZED] for this study can be found in the [https://www.jianguoyun.com/p/DTP_bNkQgbjoCxj36ZMFIAA] and GSA -gsa-human (HRA005210).


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