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Virology Journal logoLink to Virology Journal
. 2015 Aug 20;12:128. doi: 10.1186/s12985-015-0359-4

Differential expression of micrornas in porcine parvovirus infected porcine cell line

Xinqiong Li 1,#, Ling Zhu 1,2,#, Xiao Liu 1, Xiangang Sun 1, Yuanchen Zhou 1, Qiaoli Lang 1, Ping Li 1, Yuhan Cai 1, Xiaogai Qiao 1, Zhiwen Xu 1,2,
PMCID: PMC4545981  PMID: 26290078

Abstract

Background

Porcine parvovirus (PPV), a member of the Parvoviridae family, causes great economic loss in the swine industry worldwide. MicroRNAs (miRNAs) are a class of non-protein–coding genes that play many diverse and complex roles in viral infections.

Finding

Aiming to determine the impact of PPV infections on the cellular miRNAome, we used high-throughput sequencing to sequence two miRNA libraries prepared from porcine kidney 15 (PK-15) cells under normal conditions and during PPV infection. There was differential miRNA expression between the uninfected and infected cells: 65 miRNAs were upregulated and 128 miRNAs were downregulated. We detected the expression of miR-10b, miR-20a, miR-19b, miR-181a, miR-146b, miR-18a, and other previously identified immune-related miRNAs. Gene Ontology analysis and KEGG function annotations of the host target genes suggested that the miRNAs are involved in complex cellular pathways, including cellular metabolic processes, immune system processes, and gene expression.

Conclusions

These data suggest that a large group of miRNAs is expressed in PK-15 cells and that some miRNAs were altered in PPV-infected PK-15 cells. A number of microRNAs play an important role in regulating immune-related gene expression. Our findings should help with the development of new control strategies to prevent or treat PPV infections in swine.

Background

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine (Sus scrofa, ssc), where infection is characterized by early embryonic death, stillbirths, fetal death, and delayed return to estrus [1]. Additionally, PPV is associated with porcine postweaning multisystemic wasting syndrome (PMWS) and diarrhea, skin disease, and arthritis in swine [1, 2]. Even though inactivated and attenuated vaccines are widely used, the PPV-associated diseases nevertheless cause serious economic losses to the swine industry worldwide [3]. As virus replication is highly dependent on the host cell, cellular microRNA (miRNA) modification of the complex cellular regulatory networks can greatly influence viral reproduction and pathogenesis. Therefore, determining the consequences of PPV infections on cellular gene regulatory networks is urgent.

miRNAs are involved in post-transcriptional regulation of gene expression in animals, plants, and some DNA viruses. miRNAs act as regulators, inhibiting the expression of specific mRNAs by recognizing partial complementary sites in a targeted mRNA, typically within the 3’ untranslated region (3’UTR). miRNAs perform critical functions in diverse biological processes, including proliferation, apoptosis, and cell differentiation [4]. It has been well established that miRNAs play many complex roles during viral infection [5]. Therefore, an increasing number of researchers have focused on the relationship between viruses and miRNAs.

As far as we know, knowledge on the role of miRNAs in PPV infection is lacking. In this study, we detected the miRNAs expressed in porcine kidney 15 (PK-15) cells following PPV infection using high-throughput sequencing.

Methods

We used the PPV-SC-L strain, stored at the Key Laboratory of Animal Diseases and Human Health of Sichuan Province, China, in this study. PK-15 cell cultures that were 50 % confluent were infected with PPV at 10 plaque-forming units (PFU) per cell. PK-15 cells inoculated with DMEM were maintained as uninfected control cells. Cells were harvested at 24 h post-infection [6]. The cultures for each group were performed in triplicate. The infected and uninfected cells were mixed separately and used for RNA extraction. Cell viability is not affected during timecourse of infection.

Total RNA from infected PK-15 cells and normal PK-15 cells was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA quality was assessed by formaldehyde/agarose gel electrophoresis and was quantified using a ND-1000 NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, MA, USA). Approximately 20 μg total RNA was subjected to Kangcheng Bio-tech inc (Shanghai, China) for Solexa sequencing of miRNAs. The same RNA was used for qRT-PCR.

RT was performed as previously described [6]. Real-time PCR was performed using SYBR Green Real-time qPCR Master Mix (Arraystar, Rockville, MD, USA) on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The amplification conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 60 s. Table 1 lists the primers used. All samples were assayed in triplicate. The cycle threshold (Ct) values were analyzed using the 2-∆∆Ct method. The U6 gene was used as the internal control.

Table 1.

RT-qPCR primers

Gene RT primer
 U6 5’CGCTTCACGAATTTGCGTGTCAT3’
 miR-RT Primer 5’GTCGGTGTCGTGGAGTCGTTTGCAATTGCACTGGATTTTTTTTTTTTTTTTTTV3’
V = A, G, C
Gene Forward primer (5’–3’) Reversed primer (5’–3’)
 ssc-miR-10b TACCCTGTAGAACCGAATTTGT GTCGGTGTCGTGGAGTCG
 ssc-miR-30a-5p TGTAAACATCCTCGACTGGAAG GTCGGTGTCGTGGAGTCG
 ssc-miR-16 TAGCAGCACGTAAATATTGGC GTCGGTGTCGTGGAGTCG
 ssc-miR-17-5p CAAAGTGCTTACAGTGCAGGTAG GTCGGTGTCGTGGAGTCG
 ssc-miR-192 CTGACCTATGAATTGACA GTCGGTGTCGTGGAGTCG
 ssc-miR-21 TAGCTTATCAGACTGATGTTGA GTCGGTGTCGTGGAGTCG
 ssc-miR-19b TGTGCAAATCCATGCAAAAC GTCGGTGTCGTGGAGTCG
 ssc-miR-18a TAAGGTGCATCTAGTGCAGATA GTCGGTGTCGTGGAGTCG
 ssc-miR-152 TCAGTGCATGACAGAACTTGG GTCGGTGTCGTGGAGTCG
 ssc-miR-novel-chr13_10861 TTCAAGTAACCCAGGATAGGCT GTCGGTGTCGTGGAGTCG
 U6 TCGCTTTGGCAGCACCTAT AATATGGAACGCTTCGCAAA

MiRanda and TargetScan were used to predict the targets of the differentially expressed miRNAs. Predicted miRNA targets were functionally annotated through the cell component, biological process, and molecular function information supported by GO analysis. GO analysis and KEGG pathway analyses were performed using DAVID (http://david.abcc.ncifcrf.gov/) with default parameters [7].

Results

We obtained 3,575,737 and 617,535 high-quality reads from the normal and infected cell samples, respectively, remained for miRNA analysis. The length distribution of the high-quality reads ranged 16–30 nt. Most sequence reads ranged 21–24 nt, which belonged to the typical size range (Fig. 1). We identified 533 and 286 porcine miRNAs in normal PK-15 cells and infected PK-15 cells, respectively. This indicates that the normal cells contained more miRNAs than the infected cells. The change of expression of miRNAs between normal and infected PK-15 cells reflects that miRNAs can play key roles during the viral infection process, where virus can affect cellular miRNAs expression profile on their own benefit. ssc-miR-21 was the most abundantly expressed miRNA, followed by ssc-miR-30a-5p. miRNAs were considered differentially expressed when the fold change (FC) difference between groups was >2 or ≤0.5 and P ≤ 0.01, or when a miRNA was not expressed in either the infected or control group. There were 193 differentially expressed miRNAs; 128 were downregulated and 65 were upregulated. The most upregulated and downgulated miRNA were ssc-miR-10b (36-fold) and ssc-miR-18a (0.01-fold) (Table 2).

Fig. 1.

Fig. 1

Length distribution of miRNA reads from Solexa sequencing. a Adapter-trimmed reads in the infected library; b adapter-trimmed reads in the control library

Table 2.

Top 50 miRNAs significantly up- or downregulated in PK-15 cells in order of fold change (FC)

Annotation Normalized read counts length type FC Number of target genes
infected control
ssc-miR-10b 42,588 1162 22 Up 36.35 738
ssc-miR-192 3769 102 21 Up 33.74 718
ssc-miR-20a 2432 116 22 Up 19.38 1490
ssc-miR-296-3p 195 3 21 Up 15.77 1863
ssc-miR-novel-chr17-18987 195 3 19 Up 15.77 1864
ssc-miR-92b-3p 2215 133 22 Up 15.56 1757
ssc-miR-30a-5p 98,034 6320 22 Up 15.49 1147
ssc-miR-novel-chr12-7961 1886 191 22 Up 9.43 1357
ssc-miR-novel-chr14-13888 582 58 23 Up 8.71 1368
ssc-miR-34a 358 37 22 Up 7.83 1663
ssc-miR-novel-chr16-17559 55 0 22 Up 6.5 1610
ssc-miR-novel-JH11865-1-42 55 0 23 Up 6.5 1727
ssc-miR-17-5p 2868 438 23 Up 6.42 1443
ssc-miR-16 11,873 1891 22 Up 6.25 1763
ssc-miR-22-3p 2267 365 22 Up 6.07 1487
ssc-miR-146b 75 3 21 Up 6.07 1139
ssc-miR-155-5p 426 62 22 Up 6.06 1146
ssc-miR-novel-chr2-20965 52 1 23 Up 5.64 1147
ssc-miR-novel-chrx-40705 758 147 22 Up 4.89 811
ssc-miR-221-3p 758 147 22 Up 4.89 811
ssc-miR-301 114 17 23 Up 4.59 1509
ssc-miR-191 741 156 23 Up 4.52 695
ssc-miR-novel-chr6-31692 46 3 22 Up 4.30 2019
ssc-miR-181a 637 147 24 Up 4.12 1221
ssc-miR-18a 88 9541 22 Down 0.0102 995
ssc-miR-novel-chr9-37990 20 1752 23 Down 0.0170 1512
ssc-miR-novel-chr9-39041 20 1752 23 Down 0.0170 1512
ssc-miR-novel-chr6-30729 13 1317 22 Down 0.0173 1083
ssc-miR-424-5p 33 2182 22 Down 0.0196 1817
ssc-miR-31 55 3118 22 Down 0.0208 1149
ssc-miR-novel-chrX-41190 0 431 21 Down 0.0227 335
ssc-miR-novel-chr11-6750 7 547 18 Down 0.0305 1406
ssc-miR-152 332 9880 21 Down 0.0346 1161
ssc-miR-542-5p 0 277 21 Down 0.0348 732
ssc-miR-499-5p 7 472 21 Down 0.0353 974
ssc-miR-142-3p 0 238 22 Down 0.0403 887
ssc-miR-135 0 235 23 Down 0.0408 1602
ssc-miR-194a 13 541 21 Down 0.0417 842
ssc-miR-361-5p 20 704 22 Down 0.0420 867
ssc-miR-185 59 1621 22 Down 0.0423 2285
ssc-miR-193a-5p 0 201 22 Down 0.0474 1142
ssc-miR-novel-chr5-29676 0 199 23 Down 0.0478 1066
ssc-miR-183 156 3132 23 Down 0.0528 1087
ssc-miR-29c 16 366 22 Down 0.0691 1120
ssc-miR-novel-chr5-29857 42 736 19 Down 0.0697 1711
ssc-miR-29a 267 3939 23 Down 0.0701 1079
ssc-miR-19a 498 6339 23 Down 0.0800 1436
ssc-miR-19b 1161 14,587 23 Down 0.0802 1299
ssc-miR--novel-chr13_10861 169 1483 22 Down 0.1199 857
ssc-miR-21 52,611 382,830 22 Down 0.1374 789

We selected 10 miRNAs to confirm the deep sequencing data. The expression levels of ssc-miR-10b, ssc-miR-30a-5p, ssc-miR-16, ssc-miR-17-5p, and ssc-miR-192 in the PPV-infected cells were higher than in the uninfected cells, whereas ssc-miR-21, ssc-miR-19b, ssc-miR-18a, ssc-miR-152, and ssc-miR-novel-chr13_10861 were downregulated compared to the uninfected cells (Fig. 2). The results were consistent with that of the deep sequencing analysis. In addition, reverse transcription–quantitative PCR (RT-qPCR) indicated the reliability of the deep sequencing data.

Fig. 2.

Fig. 2

RT-qPCR validation and expression analysis of differentially expressed miRNAs. The relative expression levels are presented as the mean and standard deviation (SD). **P < 0.01, *P < 0.05

In our study, a total 3254 target genes were predicted for the 193 differentially expressed miRNAs. We successfully annotated about 2867 target genes through GO analysis. The upregulated biological process–related genes were involved in cellular process, metabolic process and biological regulation. The biological roles of the downregulated genes were cellular process, metabolic process, and biological regulation. GO enrichment analysis determined functional enrichment of upregulated and downregulated genes in cellular process and cell part and binding (Table 3). The target genes were classified according to Kyoto Encyclopedia of Genes and Genomes (KEGG) function annotations, and we identified pathways actively regulated by the miRNAs during PPV infection (Table 4). Some of the target genes were involved in immunity and virus infection.

Table 3.

GO analysis of swine target genes. The table shows the GO annotation of the upregulated gene (A) and downregulated gene (B) in biological process, cellular component and molecular function. Ten GO terms for each process are listed

GO.ID Term Count P-value
Biological process
 GO:0009987 cellular process 1782 1.0102E-05
 GO:0008152 metabolic process 1350 2.44953E-27
 GO:0065007 biological regulation 1260 0.000424577
 GO:0044238 primary metabolic process 1231 5.99319E-26
 GO:0044237 cellular metabolic process 1221 1.70495E-28
 GO:0050789 regulation of biological process 1192 0.002408788
 GO:0050794 regulation of cellular process 1147 0.000216533
 GO:0002376 immune system process 273 1.35305E-08
 GO:0006955 immune response 163 1.37682E-05
 GO:0000165 MAPK cascade 79 3.28195E-05
 Cellular Component
 GO:0044464 cell part 1772 1.04304E-42
 GO:0005623 cell 1772 1.25735E-42
 GO:0005622 intracellular 1589 1.48695E-38
 GO:0044424 intracellular part 1512 9.75601E-38
 GO:0043226 organelle 1258 3.15768E-22
 GO:0043229 intracellular organelle 1255 5.59497E-22
 GO:0005737 cytoplasm 1146 1.88382E-25
 GO:0043227 membrane-bounded organelle 1131 3.1329E-23
 GO:0043231 intracellular membrane-bounded organelle 1129 3.31685E-23
 GO:0044444 cytoplasmic part 886 1.59538E-15
 Molecular Function
 GO:0005488 binding 1781 7.2806E-35
 GO:0005515 protein binding 1406 1.01651E-30
 GO:0003824 catalytic activity 791 4.12422E-11
 GO:0043167 ion binding 425 2.86598E-07
 GO:0043169 cation binding 423 3.71961E-07
 GO:0046872 metal ion binding 416 4.07808E-07
 GO:0003676 nucleic acid binding 368 0.010086661
 GO:0036094 small molecule binding 366 1.33205E-09
 GO:0000166 nucleotide binding 341 4.24208E-09
 GO:0097159 organic cyclic compound binding 341 4.47117E-09
 B
 Biological Process
 GO:0009987 cellular process 1732 0.000468457
 GO:0008152 metabolic process 1280 0.000101041
 GO:0065007 biological regulation 1226 0.039474247
 GO:0044238 primary metabolic process 1179 0.011728824
 GO:0044237 cellular metabolic process 1153 0.011728824
 GO:0050789 regulation of biological process 1150 0.022891558
 GO:0050794 regulation of cellular process 1100 0.023393923
 GO:0002376 immune system process 265 3.49438E-08
 GO:0006955 immune response 161 7.5883E-06
 GO:0022402 cell cycle process 151 0.001985807
 Cellular Component
 GO:0044464 cell part 1699 1.61069E-32
 GO:0005623 cell 1699 1.89406E-32
 GO:0005622 intracellular 1506 1.8551E-26
 GO:0044424 intracellular part 1426 4.89384E-25
 GO:0043226 organelle 1212 8.24637E-20
 GO:0043229 intracellular organelle 1208 2.30659E-19
 GO:0043227 membrane-bounded organelle 1089 8.12018E-21
 GO:0043231 intracellular membrane-bounded organelle 1088 5.50768E-21
 GO:0005737 cytoplasm 1079 5.97213E-18
 GO:0044444 cytoplasmic part 836 2.48602E-11
 Molecular Function
 GO:0005488 binding 1748 3.63499E-36
 GO:0005515 protein binding 1408 1.06202E-38
 GO:0003824 catalytic activity 743 5.00132E-07
 GO:0043167 ion binding 410 2.18765E-06
 GO:0043169 cation binding 408 2.81762E-06
 GO:0046872 metal ion binding 400 4.31756E-06
 GO:0003676 nucleic acid binding 365 0.004482893
 GO:0036094 small molecule binding 335 6.20944E-06
 GO:0000166 nucleotide binding 309 2.81002E-05
 GO:0097159 organic cyclic compound binding 309 2.91504E-05

Table. 4.

Target genes of 17 differentially expressed miRNAs involved in immune response pathways

KEGG pathways Target genes Differentially expressed microRNAs FDR
T cell receptor signaling pathway CTLA4, FYN, IKBKG, NFATC2, NCK1, CD8A, PIK3CG, CDC42, PTPN6, CD4, CD40LG, ICOS, PIK3R5, MAPK14, TNF, MAP3K7 miR-10b, miR-9, miR-30a-5p, miR-17-5p, miR-16, miR-18a, miR-19b, miR-20a, miR-19a, miR-122, miR-146b, miR-55-5p, miR-181a, miR-196b, let-7 g, let-7c 8.89308E-12
Toll-like receptor signaling pathway CTSK, TLR7, MAP3K7, MAPK14, CXCL9, PIK3CG, NFKB1, CD40, STAT1, IL12B, CD86, IL6 miR-10b, miR-9, miR-30a-5p, miR-17-5p, miR-16, miR-18a, miR-19b, miR-20a, miR-21, miR-19a, miR-122, miR-146b, miR-155-5p, miR-181a, miR-196b, let-7 g, let-7c 1.04578E-07
NF-kappaB signaling pathway MAP3K7, CXCL12, DDX58, LCK, XIAP, ATM, VCAM1, NFKB1, TNF, CD40LG miR-10b, miR-9, miR-30a-5p, miR-17-5p, miR-16, miR-18a, miR-19b, miR-20a, miR-19a, miR-122, miR-146b, miR-155-5p, miR-181a, miR-196b 1.18108E-06
RIG-I-like receptor signaling pathway MAP3K7, MAPK14, DHX58, DDX58, IKBKG, TANK, IKBKB, DDX3X, NFKB1, TNF, IL12B miR-10b, miR-9, miR-30a-5p, miR-17-5p, miR-16, miR-18a, miR-19b, miR-21, miR-19a, miR-122, miR-146b, miR-155-5p, miR-181a, let-7c 1.70355E-05
Jak-STAT signaling pathway JAK2, STAT4, STAT5B, JAK3, PIK3CG, PIM1, PTPN6, TYK2, MAPK14, STAT4, STAT1, IL7R, IL12B, IL6, PIK3R5, MYC miR-9, miR-17-5p, miR-16, miR-18a, miR-19b, miR-20a, miR-21, miR-19a, miR-122, miR-146b, miR-155-5p, miR-181a, miR-196b, let-7 g, let-7c 0.000124339
NOD-like receptor signaling pathway MAP3K7, MAPK14, IKBKG, IKBKB, NFKB1, TNF, IL6 miR-10b, miR-9, miR-17-5p, miR-16, miR-18a, miR-19b, miR-19a, miR-122, miR-146b, miR-155-5p, miR-181a, let-7 g, let-7c 0.001546381

Discussion and conclusion

Previous studies have shown that viruses have evolved a wide variety of means for resisting the host immune system [810]. Furthermore, miRNAs play important roles in controlling immune regulation, including cellular differentiation and immune response [1113]. Identifying and probing miRNAs in the immune system is important for understanding their physiological and pathological roles in PPV infection. In this study, we used high-throughput sequencing to identify miRNAs.

Recent studies have provided compelling evidence that cellular miRNAs play an important role in host defense against virus infection [14]. Many immune-related miRNAs have been identified in innate and adaptive immune systems, including the miR-17—92 cluster, miR-221, miR-10, miR-196b, miR-126, miR-155, miR-150; miR-181a, miR-326, miR-142-3p, miR-424, miR-21, miR-106a, miR-223, miR-146; the let-7 family, miR-9, and miR-34 [6]. We found many differentially expressed miRNAs in the normal and PPV-infected PK-15 cells. Among them, let-7 g, miR-17-5p, miR-17-3p, miR-20a, miR-181a, miR-16, miR-146b, miR-10b, and miR-155-5p were upregulated; let-7c, miR-122, miR-18a, miR-19a, miR-19b, miR-196b, miR-21, and miR-9 were downregulated. These data suggest that viral mechanisms can affect host miRNA expression. However, we did not detect differential expression of other previously identified miRNAs (miR-223, miR-150, miR-92a), although miR-10b, miR-20a, miR-30a-5p, miR-34a, miR-17—5p, miR-16, miR-146b, and miR-155-5p expression was significantly different. In contrast, expression of the downregulated immune-related miRNAs was not significantly different, except miR-18a, miR-19b, and miR-21. This suggests that miRNAs play an important role in the coordinated regulation of immune-related gene expression in PK-15 cells in response to PPV infection.

miR-21, which had high read numbers in both normal and PPV-infected cells, was downregulated; it is related to immune response and virus replication [15]. Moreover, it is a negative regulator of toll-like receptor 4 (TLR4) signaling by targeting programmed cell death 4 (PDCD4) [16]. miR-19b and miR-18a expression was downregulated in the infected cells, suggesting that they play a negative role in PPV replication. Although viruses may downregulate host miRNA by suppressing Dicer expression, the mechanism of downregulation remains unclear [17]. Therefore, future studies are necessary for investigating the mechanism of PPV downregulation of cellular miRNA.

miR-10 expression was upregulated in the infected cells. Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), considered a target gene of miR-10, regulates the inhibitor of nuclear factor κB/nuclear factor κB (IκB/NF-κB) signaling pathway [18]. In addition, miR-10 controls brain-derived neurotrophic factor (BDNF) levels via the miRNA–mRNA regulatory network [19]. We surmise that a possible function of miR-10 in triggering an antiviral response is targeting the MAP3K7 and BDNF genes. The miR-30 family is involved in various biological and pathological processes. For example, miR-30a may be involved in B cell hyperactivity [20]. We detected miR-10 and miR-30 in this study, suggesting that they are related to the cellular immune response to PPV infection.

GO analysis showed that many of the identified miRNAs found in other studies were predicted to participate in immunity [21]. Many genes, including MAP3K7, IRAK1, TLR7, CD40, TGFBR1, RPS6KA3, IGF1R, CDC37, ITGA4, CBLB, ITGA5, IL7, ATM, DPP8, MAPK14, CD2, WNT2B, CAV1, and CD96, are involved in the immune-related programs. KEGG analysis showed that these targeted genes could participate in multiple signaling pathways, including that for retinoic acid–inducible gene-I (RIG-I)-like receptor, TLRs, Janus kinase–signal transducer and activator of transcription (JAK–STAT), and T-cell receptor. Interleukin 10 (IL10) plays an important role in virus infection by inhibiting several proinflammatory cytokines [22]. Let-7 g, let-7c, miR-19b, and miR-16 are involved in immune-related programs and may act through the target gene IL10. These results suggest that miRNAs participate in the regulation of piglet immunity. It has been established that miRNAs can target specific genes [23]; in the present study, let-7c, let-7 g, miR-18a, miR-196b, and miR-9 targeted MAP3K7, and miR-196b and miR-19b targeted dipeptidyl-peptidase 8 (DPP8), suggesting that cellular miRNAs play a key role in regulating gene expression in response to PPV infection. Genes targeted by miRNAs are involved in immune response–associated pathways in human parvovirus B19 infection [24]. We speculate that host miRNAs relate to common immune pathways in response to parvovirus infection.

To our knowledge, this is first study to survey the miRNA expression profiles in PPV-infected PK-15 cells through high-throughput sequencing. A number of miRNAs detected were previously described as immune system regulators. Target analysis confirmed that these miRNAs played an important role in PPV infection. These findings contribute to our understanding of the roles miRNAs play in host–pathogen interactions and help with the development of new control strategies to prevent or treat PPV infections in swine.

Acknowledgments

This study was supported by the Program for New Century Excellent Talents in University of Ministry of Education of China (Project No: NCET-11-1059), and by the Excellent Doctoral Dissertation Fostering Foundation of Sichuan Agricultural University (04310734). miRNA sequencing services were provided by KangChen Bio-tech, Shanghai, China.

Footnotes

Xinqiong Li and Ling Zhu contributed equally to this work.

Competing interest

The authors declare that they have no potential conflicts of interest.

Authors’ contributions

Conception and design of the experiments: XQL, LZ, ZWX, YCZ, XGS; Experimental work: XQL; PL; YHC; XGQ; QLL; Data analysis: XQL; YHC; YCZ; manuscript preparation: XQL. All authors read and approved the final manuscript.

Contributor Information

Xinqiong Li, Email: lxqll954101@163.com.

Ling Zhu, Email: abtczl72@126.com.

Xiao Liu, Email: 502401695@qq.com.

Xiangang Sun, Email: sun.xian.gang@163.com.

Yuanchen Zhou, Email: abtczyc@163.com.

Qiaoli Lang, Email: 308307923@qq.com.

Ping Li, Email: lipinga104@163.com.

Yuhan Cai, Email: 429197524@qq.com.

Xiaogai Qiao, Email: 1434143812@qq.com.

Zhiwen Xu, Phone: +86 835 2885846, Email: abtcxzw@126.com.

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