MiR-130a/2337 Targets TGF-β1 Gene To Regulate Porcine Ovarian Granulosa Cell Apoptosis

Transforming growth factor-β1(TGF-β1)is a multifunctional growth factor and one of the important ligands of the TGF-β superfamily.TGF-β1 regulates a variety of cell activities in a cell type-dependent manner,such as cell growth and differentiation,proliferation and apoptosis.TGF-β1 is highly expressed in ovarian tissue.Various cells in the ovary have the ability to express and secrete TGF-β1,such as oocytes,granulosa cells and follicular membrane cells,and TGF-β1 is detected in follicular fluid.Studies have confirmed that TGF-β1 is closely related to ovarian functions including follicular development,ovulation,steroid hormone production and granular cell status(proliferation and apoptosis),as well as female fertility.Studies have found that the expression and secretion of TGF-β1 in ovarian tissues are also regulated by a variety of factors,but there are few studies on the regulation of miRNAs.Therefore,this article intends to use the TGF-β1 gene as the research object to analyze the miRNAs regulation and mechanism of TGF-β1 gene expression in porcine ovarian granulosa cells.The research results of this article are mainly divided into the following two parts:1.miR-130 a targets TGF-β1 gene to regulate porcine ovarian granulosa cell apoptosis(1)miR-130 a targets to inhibit of TGF-β1 gene expression and function in porcine ovarian granulosa cellsThe 3’UTR sequence of TGF-β1 gene of Large White Pig was obtained by PCR amplification and sequencing,and the sequence length was 1151 bp.Sequence analysis revealed that this region contains AU enrichment elements(AREs),GU enrichment elements(GREs)and multiple miRNAs response elements(MREs)such as miR-130 a and miR-1943-3p.The RNAhybrid software predicts that miR-130 a can bind to the MRE motif(1432 nt – 1449 nt)on the 3’UTR of the TGF-β1 gene of Large White pigs.The dual luciferase test found that the overexpression of miR-130 a significantly inhibited the luciferase activity of the pig TGF-β1 gene 3’UTR wild-type reporter vector,while the luciferase activity of the MRE mutant 3’UTR reporter vector was not significant influence,indicating that TGF-β1 is the direct target gene of miR-130 a.qRT-PCR and Western blot experiments confirmed that the expression of TGF-β1 in porcine ovarian granulosa cells overexpressing miR-130 a was significantly reduced,indicating that TGF-β1 is a miR-130 a target gene in porcine ovarian granulosa cells.Flow cytometry analysis found that the apoptosis rate of granulosa cells overexpressed by miR-130 a increased significantly,and the addition of 10 ng/m L recombinant TGF-β1 could significantly reduce the effect of miR-130 a on the apoptosis of porcine granulosa cells.The above results indicate that miR-130 a induces porcine granulosa cell apoptosis by directly targeting and inhibiting the expression of TGF-β1.(2)The combined genotypes of miR-130 a and TGF-β1 mutation sites are significantly associated with reproductive traits of Large White pigsIn order to research the relationship between miR-130 a and reproductive traits of sows,the DNA mixed pool sequencing method was used to screen the miR-130 a precursor sequence and promoter region mutation sites of Large White pigs,and 2 fully linked mutation sites were found in the promoter region,namely-579G>A and-546T>C.Direct sequencing and typing found that there are 3 genotypes(GG/TT,GA/TC and AA/CC)at these 2 sites in the Large White pig population(n=325),among which GA/TC is the dominant genotype(0.537).Association analysis found that the polymorphism at the-579G>A and-546T>C sites in the promoter region of miR-130 a were had no significantly related with the reproductive traits of Large White pigs.3 mutation sites(c.1583A>G,c.1587A>G,and c.2074A>C)were found in the 3’UTR of the TGF-β1 gene of Large White pigs.The association analysis found that its polymorphisms were also no significant association related to the reproductive traits of Large White pigs.The combined genotype analysis of the Large White pig miR-130 a and TGF-β1 gene mutation sites revealed eight combined genotypes,of which the GA/TC/AA/AA/AA genotype was the dominant genotype(0.309).The results of the association between the combined genotypes and reproductive traits of Large White sows showed that litter weight(LW)of sows carried with AA/CC/AG/AG/AC genotype was significantly higher than those sows carried with GA/TC/AA/AA/AA genetype,AA/CC/AA/AA/AA genotype and GA/TC/AG/AG/AC genotype(P<0.05);the number born alive(NBA)of sows carried with AA/CC/AG/AG/AC genotype were 1 more per litter than the sows carried of GA/TC/GG/GG/CC genotype(P<0.05).In addition,with the exception of sows carried with AA/CC/AG/AG/AC genotype,the number of still born(NSB)of sows carried with GG/TT/GG/GG/CC genotype were significantly higher than that of sows carried with other genotypes(P<0.05).These results indicate that the miR-130 a gene promoter mutations and the 3’UTR mutations of TGF-β1 gene have important effects on the reproductive performance of Large White pigs.2.miR-2337 targets TGF-β1 gene to regulate porcine ovarian granulosa cell apoptosis(1)miR-2337 promotes TGF-β1 gene expression in porcine ovarian granulosa cellsBiological software analysis found that the mutations of 2 adjacent mutation sites(c.1583A>G and c.1587A>G)on the 3’UTR of TGF-β1 gene resulted in a decrease in the binding ability of miR-2337 and 3’UTR.Luciferase activity analysis found that miR-2337 can significantly inhibit the activity of wild-type pig TGF-β1 gene 3’UTR,but has no significant effect on the activity of mutant 3’UTR,indicating the mutation in the miR-2337 MRE motif affects the target inhibitory effect of miR-2337 on porcine TGF-β1 3’UTR.In porcine ovarian granulosa cells cultured in vitro,miR-2337 promote but not inhibit the expression of TGF-β1 gene mRNA and protein,and increase the concentration of TGF-β1 in the culture medium.2 miR-2337 MREs(MRE1:-621 –-604 nt and MRE2:-308 –-286 nt)were found in the promoter region of the TGF-β1 gene of Large White pigs,of which MRE1 is located in the core promoter region.Luciferase activity analysis revealed that miR-2337 can induce the up-regulation of the activity of the promoter region containing MRE1,but has no significant effect on the activity of the promoter region containing MRE2,indicating that miR-2337 is a potential small activating RNA(sa RNA)of TGF-β1 in porcine ovarian granulosa cells.The Ch IP test confirmed that miR-2337 can change the histone modification of the core promoter region of TGF-β1 in porcine ovarian granulosa cells.The enrichment of H3K4me2,H3K4me3 and H3K9 ac increases,while the enrichment of H3K9me2 decreases.The above results indicate that miR-2337 is the sa RNA of TGF-β1 in porcine ovarian granulosa cells,which activates TGF-β1 by changing histone modifications in the core promoter region.(2)miR-2337 inhibits porcine ovarian granulosa cell apoptosis by activating TGF-β1Western blot analysis found that miR-2337 can up-regulate the level of phosphorylated SMAD3(p-SMAD3,the active marker of the TGF-β signaling pathway)in porcine ovarian granulosa cells,indicating that miR-2337 is the same as TGF-β1 in porcine ovarian granulosa cells activator of TGF-β signaling pathway.Flow cytometry revealed that the overexpression of miR-2337 significantly inhibited the apoptosis rate of granulosa cells.In addition,the TGF-β receptor specific inhibitor SB431542 inactivated the TGF-β signaling pathway and inhibited the up-regulation of p-SMAD3 levels in granulosa cells induced by overexpression of miR-2337.At the same time,the inhibitor SB431542 also reversed the down-regulation of the granulosa cell apoptosis rate caused by the overexpression of miR-2337.These data indicate that miR-2337 promotes the TGF-β signaling pathway and inhibits the apoptosis of granulosa cells by activating TGF-β1 in porcine granulosa cells.In summary,this study amplified and identified the Large White pig TGF-β1 gene 3’UTR and core promoter;it was confirmed that miR-130 a and miR-2337 are inhibitors and activators of TGF-β1 in porcine ovarian granulosa cells,and directly target the 3’UTR and core promoters to inhibit and activate the expression of TGF-β1 respectively;it was found that the miR-130 a gene promoter and the 3’UTR mutation site of the TGF-β1 gene have an important impact on the reproductive performance of Large White pigs.