| As one of the most important signals transduction pathways,TGF-β/SMAD signaling pathway has been proved to regulate multiple cellular processes,such as cell growth,proliferation,differentiation,autophagy and apoptosis.Since the discovery of noncoding RNAs(mainly miRNAs and IncRNAs),the study of their regulation to various cell processes via TGF-β/SMAD signaling pathway has been a hot spot,but little is known about the knowledge of TGF-β/SMAD signaling pathway regulation to miRNAs and lncRNAs.Our previous studies have proved that TGF-β/SMAD signaling pathway plays an important role in regulating porcine granulosa cell(pGC)apoptosis and could be directly regulated by miRNAs,like miR-26b and let-7g.Based on previous RNA-Seq work,we expect to further analyze the role of noncoding RNAs in regulating TGF-β/SMAD signaling pathway and the mechanism of TGF-β/SMAD signaling pathway affects pGC apoptosis via noncoding RNAs,thus revealing the interaction between TGF-β/SMAD signaling pathway and noncoding RNAs and the role of TGF-(3/SMAD signaling pathway in regulating pGC apoptosis and follicular atresia,providing theoretical evidence for improving sow reproductive performance.The results of this study were listed as follows:1 miR-425 mediates SMAD4 feedback regulation to TGF-β/SMAD signaling pathwayFourteen differentially expressed miRNAs including 7 up-regulated miRNAs and 7 down-regulated miRNAs were identified from both RNA-seq and qRT-PCR data between the control and knockdown of SMAD4 in pGCs.Combined with mRNAs-miRNAs interaction diagram,luciferase activity and qRT-PCR analysis,we found that TGFBR2 was the common target of 4 SMAD4-inhibited miRNAs(miR-130a,miR-1306,miR-143 and miR-425).Then miR-425 was chosen for further analysis for its high inhibition efficiency.Promoter activity analysis and ChIP assay results showed that SMAD4 could bind to SBE in miR-425 promoter and inhibit miR-425 expression.Moreover,SMAD4 could positively regulate the levels of TGFBR2 and p-SMAD3 via miR-425,indicating that miR-425 mediated SMAD4 feedback regulation to TGF-(3/SMAD signaling pathway in pGCs.Apoptosis analysis showed that overexpression of miR-425 could increase pGC apoptosis and TGFBR2 overexpression could rescue miR-425-induced pGC apoptosis.In addtion,knockdown of miR-425 could dramatically inhibit pGC apoptosis and knockdown of TGFBR2 could abolish miR-425-inhibitor-induced pGC apoptosis,showing that miR-425 acted as a pro-apopotic factor and could regulate pGC apopotsis by interacting with TGF-β/SMAD signaling pathway.2 TGF-β/SMAD signaling pathway regulates pGC apoptosis through miR-143/FSHR axisOur previous study showed that FSHR was involved in porcine follicular atresia.By using RNAi and FACS,we found that knockdown of FSHR increased pGC apoptosis and inhibited FSH-induced intracellular signals(such as PKA and AKT).Bioinformatics analysis showed that FSHR is a target of miR-143.Moreover,luciferase reporter system and western blot results confirmed that miR-143 could bind to 3’-UTR of FSHR and inhibit FSHR and FSHR-induced intracellular signals expression in pGCs.Further analysis showed that miR-143 elevated pGC apoptosis by targeting FSHR.Two SBE sites were found in the miR-143 promoter,and luciferase activity analysis and ChIP assays confirmed that SMAD4 acted as a transcription factor and inhibited miR-143 expression through binding to its promoter.Knockdown of SMAD4 dramatically suppressed FSHR expression in pGCs,while overexpression of SMAD4 significantly increased FSHR expression level which could be re-inhibited by miR-143 transfection.Futhermore,in pGCs cultured in vivo,20 ng/ml TGF-β1 treatments activated TGF-β/SMAD signaling pathway and suppressed miR-143 expression and miR-143-mediated pGC apoptosis.These results prove that TGF-(3/SMAD signaling pathway could suppress pGC apoptosis via miR-143/FSHR axis in pGCs.3 LincRNA-NORFA regulates pGC apoptosis through TGF-β/SMAD signaling pathwayAt present,knowledge of IncRNAs involved in regulating mammal follicular atresia is lacking.In the previous study,we identified a highly expressed unknown transcript in pGCs and obtained its full-length sequence by using RACE.Bioinformatics analysis showed that this transcript is an intergenic noncoding RNA(lincRNA).FISH and qRT-PCR results showed that this novel lincRNA was mainly expressed in pGCs and its levels dramatically down-regulated during follicular atresia,therefore we termed it as linc-NORFA(Noncoding RNA involved in the Follicular Atresia).Further research confirmed that linc-NORFA could inhibit pGC apoptosis.Meanwhile,we found that lncRNAs could regulate its neighbor genes such as miR-126.Bioinformatics analysis showed that linc-NORFA harbored one miR-126 binding element.Luciferase activity analysis demonstrated that linc-NORFA could act as a ceRNA and sponge miR-126.FACS results showed that miR-126 acted as pro-apoptotic factor and induced pGC apoptosis mediated by linc-NORFA.Further studies showed that TGFBR2 was a functional target of miR-126,linc-NORFA/miR-126 axis regulated pGC apoptosis and TGF-β/SMAD signaling pathway through TGFBR2.Pearson correlation analysis showed that the expression levels of linc-NORFA,miR-126,and TGFBR2 had a tight relationship.In total,above observations demonstrated that linc-NORFA could regulate pGC apoptosis through TGF-β/SMAD signaling pathway.4 TGF-β/SMAD signaling pathway regulates pGC apoptosis through lnc-066/236 clusterRNA-seq data showed that 6 genes from 750Kb genomic region of porcine chromosome 9 were differentially expressed changed after SMAD4-silencing in pGCs.qRT-PCR analysis results showed that 5 of 6 genes were significantly changed when SMAD4 was knocked down or over-expressed in pGCs.Of them,two lncRNAs(lnc-066 and lnc-236)were dramatically down-regulated during porcine follicular atresia and knockdown of lnc-066 and lnc-236 could induce pGC apoptosis.Further analysis found that lnc-066 and lnc-236 could promote the expression levels of their nearby gene FZD4 and downstream β-catenin.Besides,knockdown of FZD4 could suppress FZD4/β-catenin pathway and elevate pGC apoptosis.Bioinformatics analysis showed that lnc-066 promoter contains two SBE sites,while lnc-236 promoter has three SBE sites.Luciferase activity assay and ChIP-qPCR assays shwoed that SMAD4 acted as a transcription factor and increased the expression levels of lnc-066 and lnc-236 via binding to their promoters,respectively.Moreover,TGF-β/SMAD signaling pathway could regulate pGC apoptosis through lnc-066/236 cluster/FZD4/β-catenin pathway.In summary,we confirm that miR-425 regulates pGC apoptosis through TGF-β/SMAD signaling pathway and TGF-β/SMAD signaling pathway regulates pGC apoptosis through miR-143.Meanwhile,in this article,we also prove that linc-NORFA regulates pGC apoptosis through TGF-β/SMAD signaling pathway and TGF-β/SMAD signaling pathway regulates pGC apoptosis through lnc-066/236 cluster. |
