Abstract
LncRNAs abundantly expressed in the brain have vital and wide-ranging functions in different biological processes. However, little is currently known regarding the influence of lncRNAs in developing brains after hypoxic-ischemic brain damage (HIBD). In this study, to investigate the lncRNAs expression signatures and the co-expression network of lncRNAs and mRNAs in the brain after HIBD, we established a neonatal rat HIBD model and detected the expression profiles of lncRNAs in the HIBD brain and a sham control using high-throughput sequencing. Further, highly differentially expressed lncRNAs were selected and validated by qRT-PCR. Finally, the biological functions of the selected lncRNAs were investigated by over-expressing or silencing the target genes through lentivirus transfection in hippocampal neuron cells. Our results revealed that the expression profile of lncRNAs was dramatically different between the HIBD brains and the sham control, showing as the aberrant expression of 617 lncRNA transcripts and 441 mRNA transcripts at 24 hours after HIBD. GO and KEGG analyses indicated that the differentially expressed mRNAs were mostly involved in the apoptosis signaling pathway. After validating the expression of 8 randomly selected lncRNA transcripts by qRT-PCR, we found that the TNFRSF17 gene (ID: ENSRNOG00000021987) was down-regulated in HI brains. After stable over-expression and silencing of TNFRSF17, the apoptosis rate of hippocampal neuron cells exhibited obvious changes under hypoxia or normaxia. The over-expression of TNFRSF17 could significantly up-regulate Bcl-2 but down-regulate Bax, caspase-3, and caspase-9 at the mRNA and protein levels, while the silencing of TNFRSF17 led to just the opposite phenomenon. Notably, the regulation effects of TNFRSF17 on apoptotic related genes and proteins under hypoxia were more obvious than those under normaxia. Moreover, the over-expression of TNFRSF17 reduced the apoptotic rate, but the loss of TNFRSF17 led to a high rate of apoptosis under hypoxia. Taken together, the silencing of TNFRSF17 exacerbated, while over-expression attenuated, neuron apoptosis induced by HI injury, suggesting that TNFRSF17 may be a target for the prognosis, diagnosis, and treatment of HIBD.
Keywords: High-throughput sequencing, lncRNAs, HIBD, TNFRSF17, overexpression and silencing
Introduction
Hypoxic-ischemic brain damage (HIBD), which is mainly caused by perinatal asphyxia and hypoxia, is a fatal encephalopathic disease that results in chronic neurological morbidity among neonates, with a mortality rate up to 20% [1]. Neuron apoptosis caused by ischemia anoxia plays a key role in the pathological process of HIBD because it can lead to neonatal death or permanent neurological damage [2-4]. Although significant progress on neonatal asphyxia recovery has made, its morbidity and mortality of HIBD have no noticeable improvement. Therefore, a timely intervention of apoptosis cascade can significantly reduce the neuron apoptosis.
Long non-coding RNAs (lncRNAs) are a type of mammalian genome transcript that are longer than 200 nucleotides [5]. The number of studies concerning the roles played by lncRNAs in different biological processes has increased considerably in the last decade. They can regulate specific gene expression through several mechanisms, including mediating local and higher-order epigenetic states and modulating posttranscriptional RNA processing, transport, stabilization, metabolism, and translation [6,7]. In addition, lncRNAs also maintain various cell and tissue homeostasis during physiological and pathological conditions [8,9]. For some lncRNAs, a high degree of tissue specificity has been identified. Remarkably, lncRNAs are expressed abundantly in the nervous system, and approximately 40% of lncRNAs are detected specifically in the brain [10-12]. A growing body of studies has demonstrated that lncRNAs are involved in many nervous system diseases and play critical roles in regulating the pathological processes of neurological and psychiatric diseases by maintaining neuron cellular homeostasis [13-15].
To date, however, lncRNA expression signatures and the co-expression network of lncRNAs and mRNAs in the brain after HIBD remain poorly understood. Furthermore, the function of lncRNAs in brain injury following HIBD also requires further study. Therefore, the analysis of lncRNAs may serve to broaden our understanding of the molecular mechanisms of HIBD, providing an impetus for identifying new therapeutic targets. In this study, we firstly detected the expression profiles of lncRNAs in rat HIBD brains and in a sham control using high-throughput sequencing. Further, highly differentially expressed lncRNAs were chosen according to the results of high-throughput sequencing and validated by qRT-PCR. Finally, the biological functions of the selected lncRNAs were investigated by over-expressing or silencing them through the construction of the recombinant expression lentivirus vector carrying this target gene. In this study, we aim to illustrate the lncRNAs expression signatures and the co-expression network of lncRNAs and mRNAs in the brain after HIBD and provide new therapeutic targets for HIBD intervention.
Materials and methods
High-throughput sequencing for differentially expressed mRNAs analysis
Animals and the ethics statement
Newborn male Sprague-Dawley (SD) rats (Jinling Hospital Animal Center, Nanjing, China) weighing 250-300 g were housed under environmentally controlled conditions at 23±2°C. All experimental procedures were conducted in accordance with the guidelines of the Animal Care Committee of Nanjing University and the Institutional Animal Ethics Committee (IAEC). During the experiment, every possible effort was made to minimize animal suffering.
Neonatal HIBD model
Neonatal rats were anesthetized by injecting intraperitoneally pentobarbital sodium (3%), followed by the routine disinfection of their neck skin. Then a 2~3 mm longitudinal incision was made to expose the left common carotid artery and a double-layer ligation was made. After a 2-h recovery, the rats were transferred to a sealed transparent vessel which acts as a low-oxygen chamber with a gas mixture of 8% oxygen and 92% nitrogen at a speed of 1.5 to 2.5 L/min for 2 h. The criteria for the HIBD rat model were as follows: 30 min after hypoxia ischemia (HI), brain injury symptoms such as drowsiness, burnout, and abnormal muscle tone were observed in rats. Additionally, a series of sham-operated rats were prepared. They went through exactly the same procedure as described above except that the rats in the sham control group were put on a warm pad (37°C) for 2 h but not in a low-oxygen chamber after the left common carotid artery ligation. The body temperature of all the rats was maintained at 37±0.5°C throughout the procedure using a heating pad [16].
Total RNA extraction and quality control
Twenty-four hours after HIBD, rats (n = 18) were anesthetized and perfused with 100 mL cold PBS via the ascending aorta, and then they were sacrificed. The clean hippocampus tissues were quickly cut into small pieces, rinsed with PBS and dissolved with TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). The lysates from three rats in each group were pooled into one sample as previously described [17]. The concentration and quality of the RNA and the RNA integrity were evaluated. Each sample had an RIN above 7.0. After removal of the rRNA, the purified RNAs were fragmented to approximately 200 bps and then were amplified and transcribed into fluorescent cRNA using the TruSeq® RNA LT/HT Sample Prep Kit (Illumina, Santiago, CA, USA). The purified library products were evaluated. Each of the purified RNA samples showed an A260:A230 ratio above 2.0 and an A260:A280 ratio above 1.8, suggesting that the RNAs were sufficiently pure for RNA-seq.
RNA-seq and data analysis
The purified library products were diluted to 10 pM and then subjected to RNA-seq using an Illumina HiSeqTM 2500 (Illumina, Santiago, CA, USA) at Guangzhou RiboBio Co., Ltd. Paired-end reads were aligned to the rat transcriptome with Tophat2 as previously described [18]. RNAseq data were normalized for GC (guanine-cytosine) content with EDASeq software. RPKM (reads per kilo base per million) values were used to quantify the expression levels of lncRNAs and mRNAs by normalizing for lncRNA or mRNA length and library size and were log transformed using log2 (RPKM + 0.01) [19]. Differential expression was determined with DEGseq software, and Q-value was used to denote the significance of the P-value (q-value < 0.05 is recommended) [20].
Bioinformatics analysis
All differentially expressed mRNAs were selected for Gene Ontolo-gy (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to investigate the potential role of the lncRNAs co-expressed with mRNAs. A GO analysis was performed with KOBAS 2.0 software. The false discovery rate (FDR) was used to denote the significance of the P-value (FDR < 0.05 is recommended). The differentially expressed mRNAs and the enrichment of different pathways were mapped using the KEGG pathways with KOBAS 2.0 software (http://www.genome.jp/kegg) [21,22]. The significance of the KEGG pathways among differentially expressed genes was denoted by the FDR.
Validation for gene expression of the differentially expressed mRNAs by qRT-PCR
To identify the veracity of the sequencing, we randomly selected eight lncRNA transcripts for validation. The total RNA extracted from the control or HIBD brains was reversely transcribed to cDNA. The expression levels of these eight lncRNAs were further assessed by qRT-PCR using iQ TM SYBR® Green Supermix (Bio-Rad Laboratories, Inc. Hercules, CA, USA).
The effects of the TNFRSF17 gene on hippocampal neuron cells under hypoxia and normaxia
The cultivation of rat hippocampal neuron cells
The hippocampi of the E18 fetal rat pups were dissected by gently exfoliating the vessels around the hippocampus on a dish with DMEM + 10% FBS (Gibco, Carlsbad, CA, USA). The long axis of the hippocampus was cut into 1 mm3 pieces and digested with 3 mL fresh papaya enzyme and DNA enzyme (2 mg.mL-1) for 20-30 min at 37°C. Aspirate off supernatant, the digested blocks were added with 1.5 mL DMEM medium containing 10% FBS and subjected to gentle blowing 10 times. After counting the cells, the supernatant was transferred to a 6-well plate coated with poly-D-lysine at a density of 5ore6 cells/well. Six hours after plating,a culture medium was exchanged for a neurobasal medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 2% B27, 0.5 mM Glutamine, and 1% penicillin/streptomycin, and the cells were maintained in a humidified 37°C atmosphere containing 5% CO2.
Transfection of pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA lentivirus vector
For the over-expression or the silencing of the tumor necrosis factor receptor superfamily member 17 (TNFRSF17) in hippocampal neuron cells, TNFRSF17 primers and TNFRSF17 mRNA were respectively designed and synthesized by Sangon Biotech Co., Ltd. On the one hand, TNFRSF17 primers were obtained for TNFRSF17 gene amplification. The amplified DNA products were double digested by the restriction endonucleases EcoR1 and BamH1, and then linked to the pEGFP-N1 vector. The recombinant plasmid (5 μl) was used to transform DH5α competent cells (BioVector NTCC Inc.). On the other hand, three target sequences or the scramble sequence (listed as Table 1) for TNFRSF17 mRNA were chosen according to the RNAi Consortium (TRC) shRNA Library for TNFRSF17 silencing. Double-stranded oligonucleotides were produced by mixing the corresponding forward and reverse primers and annealing at 95°C for 5 minutes. Then, they were inserted into a pLKO.1 lentivirus vector between the Age I and EcoR I (NEB, USA) restriction sites. After treatment by T4 DNA ligase, the recombinant vector was transformed into E.coli DH5α competent cells for plasmid amplification. The extracted plasmids from the positive colonies were sequenced by Decode Genomics Biotechnology Co., Ltd to identify the target sequences. The hippocampal neuron cells were prepared as described above. The transfections of pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA were performed in 293T cells with MIO = 1:100 according to the instruction manual of the ViraDuctin™ Lentivirus Transduction Kit (Cell Biolabs, San Diego CA USA). After 72 h incubation in 5% CO2 incubator, transfection was terminated for a follow-up study.
Table 1.
The TNFRSF17 shRNA single-strand oligo nucleotides
| Name | F or R | Sequence |
|---|---|---|
| shRNA1 | Forward | 5’-CCG GGA GGA CTG TGT CAA GAG CAA ACT CGA GTT TGC TCT TGA CAC AGT CCT CTT TTT G-3’ |
| Reverse | 3’-AAT TCA AAA AGA GGA CTG TGT CAA GAG CAA ACT CGA GTT TGC TCT TGA CAC AGT CCT C-5’ | |
| shRNA2 | Forward | 5’-CCG GCG GGT GAC TAC GGC AAG TCA ACT CGA GTT GAC TTG CCG TAG TCA CCC GTT TTT G-3’ |
| Reverse | 3’-AAT TCA AAA ACG GGT GAC TAC GGC AAG TCA ACT CGA GTT GAC TTG CCG TAG TCA CCC G-5’ | |
| shRNA3 | Forward | 5’-CCG GGT TCA GTG AAA GGG ACG TAC ACT CGA GTG TAC GTC CCT TTC ACT GAA CTT TTT G-3’ |
| Reverse | 3’-AAT TCA AAA AGT TCA GTG AAA GGG ACG TAC ACT CGA GTG TAC GTC CCT TTC ACT GAA C-5’ | |
| Scramble | Forward | 5’-CCG GGA GGA CTG CCG CAA TAT CAA ACT CGA CCC TGC TCA TGA CAC AGT CCT CTT TTT G-3’ |
| Reverse | 3’-AAT TCA AAA AGA CCA CTG TGT CCC GAG CCA ACG CGA GTT TTC TCT TGA TCC AGT CCT C-5’ |
qPCR assay for the expression of TNFRSF17 and apoptotic related genes
To identify the silencing and over-expression efficiency of the lentivirus and the influence of TNFRSF17 on the apoptosis signaling pathway, total RNA was extracted from each group of hippocampal neuron cells under hypoxia or normaxia at 72 hours post transfection. Then, cDNA was acquired using HiFiScript cDNA Synthesis Kit (ComWin Biotech Co., Ltd, Jiangsu, China). The expression levels of the TNFRSF17 gene and apoptotic related genes were examined by qRT-PCR, as described above. The 2-ΔΔCt method was used for data analysis. All samples were normalized to the expression of GAPDH. The detailed primer information is available in Table 2.
Table 2.
The detailed primer information for apoptotic related genes
| Name | F or R | Sequence |
|---|---|---|
| Bcl-2 | Forward | 5’-CCG GGA GAT CGT GAT GAA GT-3’ |
| Reverse | 5’-ATC CCA GCC TCC GTT ATC CT-3’ | |
| Bax | Forward | 5’-CCA AGA AGC TGA GCG AGT GTC-3’ |
| Reverse | 5’-TGA GGA CTC CAG CCA CAA AGA-3’ | |
| Caspase-3 | Forward | 5’-CAT GGA AGC AAG TCG ATG GA-3’ |
| Reverse | 3’-CGG CAG TAG TCG CCT CTG A-5’ | |
| Caspase-9 | Forward | 5’-CCA CTG CCT CAT CAT CAA CA-3’ |
| Reverse | 3’-CCT GGT ATG GGA CAG CAT CT-5’ | |
| GAPDH | Forward | 5’-CAA GTT CAA CGG CAC AGT CAA-3’ |
| Reverse | 3’-CTC AGA TGA CCG CAG AAG TGG T-5’ |
Western blot analysis for the expression levels of apoptotic related proteins
To investigate the effects of TNFRSF17 on the expression of apoptotic related proteins in hippocampal neurons under hypoxia or normaxia, cells were collected and lysed in a lysis buffer at 4°C for 60 min. Equal amounts of protein were separated by SDS-PAGE and then electro-transferred onto a polyvinylidene difluoride membrane (Millipore Bedford, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with corresponding primary antibodies at 4°C overnight and secondary antibodies for 2 h. Densitometry analysis of protein bands were done by Image J2 software. The experiment was repeated three times.
Annexin V-FITC/PI double-staining for apoptosis analysis
Hippocampal neuron cells under hypoxia or normaxia were respectively transfected with a pEGFP-N1-TNFRSF17 or pLKO.1-TNFRSF17 shRNA lentivirus vector, harvested 24 hours post transfection, and then treated by an Annexin V/pyridine iodide (PI) apoptosis detection kit (Beyotime Biotechnology, Nanjing, China). Cell suspension at density of 1×108 cells/mL was mixed with fluorescein isothiocyanate labeled Annexin V and PI, and then analyzed by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA). The blank control and single-stained cells were used to set the gate in flow cytometry. The experiment was repeated three times.
Statistical analysis
All data are presented as the mean ± SEM unless otherwise stated. Comparisons between the two groups were analyzed using the Student’s t-test. P < 0.05 indicated that the difference was statistically significant.
Results
Some differentially expressed mRNAs were found in HIBD rat brains by GO and KEGG analysis
The function of lncRNAs is thought to be reflected in their associated protein-coding genes [7]. To clarify the potential function of these differentially expressed mRNA-related lncRNAs, GO enrichment analysis of differentially expressed mRNAs was used. Three domains-biological processes, cellular components and molecular functions of GO enrichment analysis were investigated. The significance of GO analysis in each domain was denoted by FDR (FDR < 0.05 is recommended), as showed in Figure 1A. The results indicated a remarkable difference in lncRNA and mRNA transcripts between the HIBD and control brains. Approximately 617 lncRNA transcripts and 441 mRNA transcripts were aberrantly expressed in the hippocampus tissues of neonatal rats at 24 hours after HIBD. Here, the up-regulated genes were marked as red, while the down-regulated genes were denoted by blue. Notably, the response to the wounding, the immune system process, and the inflammatory response were the most enriched terms in biological processes (BP), showing as the up-regulation of Il7r (ID: ENSRNOG00000058446), LRRK2 (ID: ENSRNOG00000004 048), Ttf2 (ID: ENSRNOG00000057761) and the down-regulation of Tnfrsf17 (ID: ENSRNOG00000021987). The extracellular region parts and the extracellular space were the most enriched terms in the cellular components (CC), showing as the up-regulation of Hsph1 (ID: ENSRNOG00000000902) and Urm1 (ID: ENSRNOG00000026636). Protein binding, binding, and chemokine activity were the most enriched terms in the molecular functions (MF), showing as the up-regulation of Zic1 (ID: ENSRNOG00000014644) and Atp2a1 (ID: ENSRNOG00000047124), as showed in Figure 1D. Moreover, to reveal the key lncRNAs and their potential functions in HIBD, we constructed a lncRNA/mRNA co-expression network and investigated the potential interactions between the lncRNA transcripts and the mRNA transcripts. More than 1,800 network nodes were composed. Ten network nodes with high correlation (COR)-values were selected, and the co-expression network was established using Cytoscape software. Similarly, most of the lncRNA-co-expressed mRNA transcripts were involved in the inflammatory response (the biological process), the extracellular region (the cellular component) and protein binding (the molecular function) according to GO enrichment. In addition, as shown in Figure 1B, the KEGG pathway analysis indicated that the most enriched pathways involving significant differentially expressed mRNAs were the ether lipid metabolism pathway (downstream), the p53 and B cell receptor signaling pathway, progesterone-mediated oocyte maturation, the cell cycle and tumor necrosis factor (TNF) signaling pathway (upstream). By comparing the abundance of mRNAs between the HIBD and control rats, nine mRNAs that are differentially expressed (P < 0.05) in the hippocampus were identified (Figure 1C). In addition, for the absolute values of logFC, the majority of differentially expressed mRNAs exhibit a 1- to 8-fold difference, and 2 mRNAs showed differences greater than 4-fold between HIBD and normal rat brains.
Figure 1.
The detection of differentially expressed mRNAs in HIBD rat brains by high-throughput sequencing. A. Scatter plot of the high-throughput sequencing data that present the gene expression levels of Control vs HIBD. The up-regulated genes were marked as red, while the down-regulated genes were denoted by blue. FDR < 0.05 is recommended. B. The enriched pathways of differentially expressed mRNAs according to GO and KEGG analyses. The rich factor represents the degree of enrichment. Dot size represents the number of significantly differentially expressed mRNAs. C. The MA plot on the left and the volcano plot on the right. The differentially expressed mRNAs are graphed on the scatter plot to visualize variations in mRNA expression between HIBD and normal rat brains. Diagrams reflect fold change value (HIBD/Control) distribution in the differentially expressed mRNA numbers. In MA and volcano plots, red dots represent the differentially expressed miRNAs, whereas black represents miRNAs with similar expressions. D. The hierarchical clustering of differentially expressed mRNAs. Left: Control group; Right: HIBD model group. Red represents high expression, green designates low expression. E. Validation of gene expression of the differentially expressed mRNAs by qRT-PCR. The qRT-PCR results confirm the accuracy of RNA-seq data. □ are the sequencing data; ■ are the qRT-PCR results.
The expression levels of differentially expressed mRNAs from RNA-seq could be validated by qRT-PCR
To validate the RNA-seq data, qRT-PCR was performed using eight randomly selected lncRNAs (5 upregulated and 3 downregulated). The qRT-PCR analysis revealed that the expression levels of ENSRNOG00000047124 (gene name: Atp2a1), ENSRNOG0000 0058446 (gene name: Il7r), ENSRNOG00000004048 (gene name: Lrrk2), ENSRNOG 00000014644 (gene name: Zic1) and ENSRNOG00000057761 (gene name: Ttf2) were upregulated, while those of ENSRNOG00000021987 (gene name: Tnfrsf17), ENSRN OG00000026636 (gene name: Urm1) and ENSRNOG00000025889 (gene name: Gnas) were downregulated in HOBD rat brains. The qRT-PCR results confirmed the accuracy of RNA-seq, as Figure 1E shown.
TNFRSF17 gene attenuated hypoxia-induced apoptosis of hippocampal neuron cells by interfering with apoptosis signaling pathway
Transfection of pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA lentivirus vector led to the over-expression and silencing of TNFRSF17 in hippocampal neuron cells
The hippocampus is susceptible to HIBD injury [23]. Therefore, we obtained the hippocampi from E18 fetal rat pups in this study to investigate the effects of the TNFRSF17 gene on hippocampal neuron cells under hypoxia or normaxia. After the transfection of pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA lentivirus vector, the expression levels of TNFRSF17 gene were detected by qRT-PCR. The results showed that the transfection of pEGFP-N1-TNFRSF17 significantly up-regulated TNFRSF17 gene expression with an increased production rate of nearly more than five times as compared with pEGFP-N1 empty vector treated group (P < 0.01, vs NC group), Figure 2A. Contrarily, the transfection of the pLKO.1-TNFRSF17 shRNA lentivirus vector could down-regulate TNFRSF17 gene expression with a production rate almost three times lower compared with the pLKO.1-scramble shRNA treated group (P < 0.01, vs sh-NC group, Figure 3A), suggesting a stable over-expression or silencing of the TNFRSF17 gene in hippocampal neuron cells.
Figure 2.
The effects of over-expressed TNFRSF17 on regulating the expression of apoptotic related genes and proteins under normaxia or hypoxia. The expression levels of TNFRSF17 in hippocampal neuron cells after the transfection of the pEGFP-N1-TNFRSF17 lentivirus vector (A). The expression levels of apoptotic related genes from qRT-PCR analysis (B) and apoptotic related protein from Western blot (C). The histograms show the relative band intensity ratio generated from three independent experiments (D). crtl: cells without treatment; NC: cells transfected with pEGFP-N1 empty vector; TNFRSF17: cells transfected with pEGFP-N1-TNFRSF17. **P < 0.01 compared with NC group.
Figure 3.
The effects of silencing TNFRSF17 on regulating the expression of apoptotic related genes and proteins under normaxia or hypoxia. The expression levels of TNFRSF17 in hippocampal neuron cells after the transfection of pLKO.1-TNFRSF17 shRNA lentivirus vector (A). The expression levels of apoptotic related genes from qRT-PCR analysis (B) and apoptotic related protein from western blot (C). The histograms show the relative band intensity ratio generated from three independent experiments (D). crtl: cells without treatment; sh-NC: cells transfected with pLKO.1-scramble shRNA; sh-TNFRSF17: cells transfected with pLKO.1-TNFRSF17 shRNA. **P < 0.01 compared with sh-NC group.
TNFRSF17 mediated the expression of apoptotic related genes and proteins
As the results of the KEGG analysis show, the most enriched pathways involving significant differentially expressed mRNAs were the apoptosis and B cell receptor signaling pathways. In the apoptosis signaling pathway, some genes (Bcl2, Bax, caspase-3 and caspase-9) and the proteins encoded by these genes play a vital role in cell apoptosis. Therefore, in the present study, we constructed the pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA lentivirus vectors to clarify the effects of TNFRSF17 on the expression of apoptotic related genes and proteins in hippocampal neuron cells under hypoxia or normaxia. As shown in Figure 2B, TNFRSF17 over-expression significantly up-regulated Bcl-2 expression level but down-regulated the expression levels of Bax, caspase-3 and caspase-9 at the mRNA levels under either hypoxia or normaxia (P < 0.01, vs NC group). Meanwhile, there was no significant difference of over-expressed TNFRSF17 on apoptotic related genes between the hypoxia and normaxia treated cells. Contrarily, in the sh-TNFRSF17 group, in which TNFRSF17 was silenced, the expression level of Bcl-2 was distinctly down-regulated while the expression levels of Bax, caspase-3, and caspase-9 were obviously up-regulated, as Figure 3B showed. Notably, the decreased production rates of Bax, caspase-3 and caspase-9 under hypoxia were more obvious than those of normaxia groups. Similarly, the corresponding protein levels were also changed in accordance with the mRNA levels (Figures 2C and 3C). It suggests that TNFRSF17 can up-regulate Bcl-2, while it suppresses the expression levels of Bax, caspase-3, and caspase-9. The down-regulation of TNFRSF17 induced by hypoxia may lead to an increased expression of Bax, caspase-3 and caspase-9.
TNFRSF17 attenuated hypoxia-induced apoptosis of hippocampal neuron cells
In order to further confirm the effects of TNFRSF17 on the hypoxia-induced apoptosis of hippocampal neuron cells, the Annexin V-FITC/PI double-staining method was used. As shown in Figure 4, in cells transfected with sh-TNFRSF17, a high apoptotic rate was observed under both hypoxia and normaxia (P < 0.01, vs sh-NC group). On the other hand, the over-expression of TNFRSF17 significantly decreased the apoptotic rate in hippocampal neuron cells. Importantly, the rates of apoptosis under hypoxia were always greater than they were under normaxia. From these data, we confirmed that TNFRSF17 plays an important role in regulating cell apoptosis, and the down-regulation of TNFRSF17 induced by hypoxia may aggravate neuron apoptosis.
Figure 4.
The effects of TNFRSF17 on hypoxia-induced apoptosis of hippocampal neuron cells. Flow cytometry analysis for the cell apoptosis in hippocampal neuron cells with TNFRSF17 over-expression (A) or TNFRSF17 silencing (C) under hypoxia or normaxia. The quantification of cells apoptotic rate when TNFRSF17 was over-expressed (B) or silenced (D) under hypoxia or normaxia. crtl: cells without treatment; sh-NC: cells transfected with pLKO.1-scramble shRNA; sh-TNFRSF17: cells transfected with pLKO.1-TNFRSF17 shRNA; NC: cells transfected with pEGFP-N1 empty vector; TNFRSF17: cells transfected with pEGFP-N1-TNFRSF17. **P < 0.01 compared with sh-NC group or NC group.
Discussion
LncRNAs abundantly expressed in the brain have vital and wide-ranging functions in different biological processes. They are involved in various nervous system diseases and have been implicated as regulators of the pathological processes of neurological and psychiatric diseases by regulating the cell cycle, chromatin structure, and RNA stability [13-15]. Previous studies in the adult rat have shown that after a stroke, cerebral lncRNA expression profiles are extensively altered and contribute to the stabilization of mRNA expression [24]. Few studies to date, however, have evaluated lncRNA changes in the developing brain after HIBD injury. The purpose of this study was to search for genes unique in developing rat brains to normaxia or hypoxia that explain the observed hypoxic-ischemic injury on neurons. Firstly, we evaluated the cerebral expression of lncRNAs in both HIBD-injured neonatal rats and sham controls using high-throughput sequencing analysis. The data of RNA-seq were quantified by RPKM (reads per kilo base per million) values, which is the recommended and most common method to estimate the level of gene expression [25]. We found that, approximately 617 lncRNA transcripts and 441 mRNA transcripts which were aberrantly expressed in the hippocampus tissues of neonatal rats were detected at 24 hours after HIBD. It suggests that there exist a large number of lncRNA changes in the developing brain after HIBD injury. The differentially expressed mRNAs were then subjected to GO and KEGG pathway analyses to investigate the potential role of the lncRNAs co-expressed with mRNAs. Here, the GO analysis, which covers three domains: the cellular component, the molecular function, and the biological process [26], was used to provide a label classification of gene function and gene product attributes (http://www.geneontology.org). KEGG pathways assay was performed to map the enrichment of the different pathways of the differentially expressed mRNAs. Our results revealed that the terms most prevalent among differentially expressed genes in HIBD are the response to wounding, the immune system process and the inflammatory response, protein binding, binding and chemokine activity. This particular category includes 9 genes, including Il7r and Tnfrsf17, etc. (Figure 1D). In particular, 8 randomly selected lncRNAs (5 upregulated and 3 downregulated) were validated by RT-qPCR, and the results were similar to the sequence data. Collectively, these findings showed that hippocampal HIBD injury influenced lncRNAs expression in neonatal rats and these altered lncRNA expressions might exert effects on the development and progress of HIBD via regulating mRNA expression.
The novel findings of RNA-seq and qRT-RCR resulted in the identification of at least eight candidate genes that warrant further investigation as to their role in HIBD. Among them, TNFRSF17, mainly expressed in mature B cells, can maintain B-cell development and immune response, and directly combine with cytokine BAFF to activate the NF-kB and MAPK/JNK pathways [27]. Notably, TNFRSF17 combines with TRAF family members to induce cell apoptosis and proliferation [28]. In some studies, it was reported that TNFRSF17 can promote cell apoptosis by the T-cell dependent activation of memory B cells [29]. Neuron apoptosis, which is the main cause of death, plays a key role in the pathological process of HIBD because it can lead to neonatal death or permanent neurological damage [2-4]. Although significant progress on neonatal asphyxia recovery has been made, the morbidity and mortality of HIBD have no obvious improvement. Therefore, a timely intervention of the apoptosis cascade can significantly reduce neuron apoptosis. A KEGG analysis demonstrated that the most enriched pathways involving significant differentially expressed mRNAs were the apoptosis and B cell receptor signaling pathways. In the apoptosis signaling pathway, some genes (Bcl2, Bax, caspase-3 and caspase-9) and the proteins encoded by these genes play a vital role in cell apoptosis. In this study, we speculated that TNFRSF17 may play a key role in HIBD by regulating the cell apoptosis of neurons. To clarify the effects of TNFRSF17 on the expression of apoptotic related genes and proteins in neuron under hypoxia or normaxia, we obtained the hippocampal neuron cells from E18 fetal rat pups and transfected the pEGFP-N1-TNFRSF17 and pLKO.1-TNFRSF17 shRNA lentivirus vectors into cells to over-express or silence TNFRSF17. The results demonstrated that over-expressed TNFRSF17 significantly up-regulated Bcl-2 but down-regulated Bax, caspase-3, and caspase-9 at the mRNA and protein levels under either hypoxia or normaxia (P < 0.01, vs NC group or sh-RNA group). However, after TNFRSF17 silencing, the expression level of Bcl-2 was distinctly down-regulated, while Bax, caspase-3 and caspase-9 were obviously up-regulated, as Figure 3B shows. Notably, the regulation effects of TNFRSF17 on apoptotic related genes and proteins under hypoxia were more obvious than they were under normaxia. Then Annexin V-FITC/PI double-staining was performed to further confirm the effects of TNFRSF17 on hypoxia-induced apoptosis in hippocampal neuron cells. Similarly, the over-expression of TNFRSF17 reduced the apoptotic rate, while the loss of TNFRSF17 led to a high rates of apoptosis under hypoxia. Taken together, we confirmed that TNFRSF17 plays an important role in neuron apoptosis, and the down-regulation of TNFRSF17 induced by hypoxia may aggravate neuron apoptosis.
In conclusion, our study demonstrated that HI injury alters the expression profiles of lncRNAs in neonatal rat brains. Particularly, ENSRNOG00000021987 is down-regulated in HI brains. The over-expression of lncRNA ENSRNOG00000021987 is attenuated, while silencing exacerbates cell apoptosis in hippocampal neuron cells subjected to hypoxia stimulation. These findings could help enrich our knowledge of the pathogenesis of and provide new therapeutic targets for HIBD.
Acknowledgements
This work was supported by the Clinical Advanced Techniques, Primary Research & Development Plan of Jiangsu Province (BE2017719), the Pediatric Medical Innovation Team of Jiangsu Province (CXTDA2017022), the National Youth Fund (81601355), and the Postdoctoral Fund of Jiangsu Province (1701162C).
Disclosure of conflict of interest
None.
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