Construction Of Gene Regulatory Networks From Porcine Ovarian Whole Transcriptomic Data And Characterization Of Some Key Gene-gene Interactions

Fecundity of sows is considered the most important economic trait as it is critical for swine farm profitability.However,there are considerable differences in fecundity rates among individual pigs,improving sow fecundity is extremely important for swine producers.Reproductive regulation is a complex process which is controlled by multiple organs.The ovary is the most important reproductive organ of sows and is responsible for synthesizing and secreting hormones,which are essential for the maintenance of the normal reproductive cycles and hormone levels.Ovarian folliculogenesis,ovulation and formation and regression of the corpus luteum occur in the ovary,which repeatedly take place over the reproductive life and regulate reproduction in mammals.Recent studies have demonstrated that non-coding RNAs?lncRNA,circRNA and miRNA?play a significant role in the development of ovaries.Although a previous report described miRNA expression profiles in pig ovarian tissues correlating with fecundity,much less is known about the regulatory molecular mechanisms of fecundity in sows.In this study,by using different types of RNA libraries?RNA-seq and small RNA-seq?,we investigated the complete transcriptome of ovarian tissue during the luteal?L?and follicular phases?F?of the estrous cycle in Large White pigs with high?H?and low fecundity?L?.We aimed to identify potential regulators?lncRNAs,circRNAs and miRNAs?of fecundity in pigs.Moreover.we explored the regulatory mechanism between TCONS₀0814106 and ssc-miR-1343 and between TGFBR1 and ssc-miR-1343 by using dual luciferase reporter assay,overexpression,RNA interference?RNAi?,and Western Blot.The main study outcomes were as follows:Total number of piglets born?TNB?of 590 multiparous Canadian Large White cyclic sows were calculated,eight sows with similar parity from high?H?and low?L?fecundity were chosen for the study?n=8 for the H group and n=8 for the L group?.Ovarian tissues were obtained on day 14?day 1=first day of estrus?after estrus,as in the luteal phase?n=4 for H fecundity sows during the F phase[FH]and n=4 for L fecundity sows during the F phase[FL]?.and on day 20 of the estrous cycle,as in the follicular phase?n=4 for H fecundity sows during the F phase[FH]and n=4 for L fecundity sows during the F phase[FL]?.Whole transcriptomic data were generated from 16 ovarian tissues.A total,24,447 lncRNA,21,386 circRNA,27,370 mRNAs transcripts,216 known miRNAs and 1724 novel miRNAs were identified in this study.A total of 457?161 up-regulated and 296 down-regulated?mRNAs,956?345 up-regulated and 611 down-regulated?lncRNAs,1079?458 up-regulated and 621 down-regulated?circRNAs transcripts,and 122?68 up-regulated and 54 down-regulated?miRNAs were differentially expressed in LH vs.LL?P<0.05?.Furthermore,pathway enrichment analyses were performed for differentially expressed genes and the potential target genes of non-coding RNAs?lncRNA,circRNA and miRNA?.We noticed that multiple pathways were closely involved in the reproductive process,such as steroid biosynthesis,lysosome,hippo signaling pathway,ovarian steroidogenesis,foxO signaling pathway,GnRH signaling pathway,PI3K-Akt signaling pathway,Ras signaling pathway,Cytokine-cytokine receptor interaction,MAPK signaling pathway,Estrogen signaling pathway and Jak-STAT signaling pathway.A total of 475?253 up-regulated and 222 down-regulated?mRNAs,415?247 up-regulated and 168 down-regulated?lncRNAs,1077?347 up-regulated and 730 down-regulated?circRNAs transcripts,and 46?32 up-regulated and 14 down-regulated?miRNAs were differentially expressed in FH vs.FL?P<0.05?.Furthermore,pathway enrichment analyses were performed for differentially expressed genes and the potential target genes of non-coding RNAs?lncRNA,circRNA and miRNA?.We noticed that multiple pathways were closely involved in the reproductive process,such as Notch signaling pathway,Steroid biosynthesis,Retinol metabolism,TGF-beta signaling pathway,cell cycle and Wnt signaling pathway.Based on bioinformatics analysis,the lncRNA-miRNA,circRNA-miRNA and miRNA-mRNA interactions were integrated to establish competing endogenous RNA?ceRNA?networks.In the lncRNA-miRNA-mRNA networks,we observed that miR-1343 and miR-1307 had the most interactions with lncRNA and mRNA?LH vs.LL?.In the FH vs.FL comparison,we observed that miR-671-5p had the most interactions with lncRNA and mRNA,implying it was the hub gene in the network.The reverse transcription real-time quantitative PCR?RT-qPCR?results validated and supported these results,such as TCONS₀0814106,TCONS₀0521721,miR-1343,TGFBR1and ILF3.The primary granulose cells?GCs?were treated with Follicle stimulating hormone?FSH,10 IU/mL?and human chorionic gonadotropin?hCG,5 IU/mL?for 24 h,respectively.The RT-qPCR results revealed that FSH and hCG could significantly increase TCONS₀0814106 expression levels.These findings demonstrated that TCONS₀0814106 expression was regulated by FSH or hCG,indicating that TCONS₀0814106 is involved in reproductive processes.The dual luciferase assay was used to validate the targeting relationship among the key nodes in the regulatory network,such as TCONS₀0814106,miR-1343 and TGFBR1.The results showed that overexpression of miR-1343 significantly reduced the relative luciferase activity of wild-type TCONS₀0814106 and TGFBR1 3'-UTR reporter genes in 293 T cells,but had no dramatically effect on luciferase activity of mutant-type luciferase reporter.Moreover,RT-qPCR and Western blot?WB?analysis showed that the expression of TCONS₀0814106 and TGFBR1 was reduced by miR-1343,In contrary,a significant increase of expression level was observed when transfected with pLV?Exp?-miR-1343.These results proved that TCONS₀0814106 and TGFBR1 are target genes of miR-1343.Cell counting kit-8?CCK-8?and apoptosis assay showed that overexpression of TCONS₀0814106 leads to an increase of cell proliferation capacity with a decrease of apoptosis rate in GCs.Not surprisingly,miR-1343 could abolish these effects caused by TCONS₀0814106 in GCs.In contrast,knockdown of TCONS₀0814106 results in reduced GC proliferation and increased GC apoptosis rate.As expected,inhibition of miR-1343 could visibly reverse the effects of TCONS₀0814106 knockdown on the proliferation and apoptosis of GCs.In addition,RT-qPCR and western blot results showed that overexpression of TCONS₀0814106 markedly increased the mRNA and protein expression of TGFBR1 in porcine GCs.And it was reversed by co-transfection with pLV?Exp?-TCONS₀0814106 and pLV-sh-miR-1343.In contrast,knockdown of TCONS₀0814106 significantly decreased the mRNA and protein expression of TGFBR1 in GCs.We co-transfected pLV-sh-TCONS₀0814106 and pLV?Exp?-miR-1343 into GCs,and found that the repression was reversed.In summary,we successfully performed the ovarian whole transcriptomic profiling of Large White sows with high and low fecundity to identify the differentially expressed ncRNAs closely involved in the reproductive process.In addition,the fecundity regulation networks were constructed;We also found that TCONS₀0814106 and TGFBR1 are target genes of miR-1343.TCONS₀0814106 reinforced proliferation and repressed apoptosis,which could be reversed by miR-1343 in porcine GCs.In addition,TCONS₀0814106 could serve as ceRNA to regulate the expression of TGFBR1.