The Mechanism Of Regulation Of CYP19A1 Gene Expression In Porcine Granulosa Cells

China is a great pig-raising country,but the low reproductive capacity becomes one of the bottlenecks that restricts the development of pig-raising industry.Ovulation is the initial factor that determines the reproductive potential.It was reported that sows with higher ovulation had less folliclular atresia rate,low E2 concentration is one of the main features of atresia.E2 is necessary to maintain productive process of pig and appropriate concentration of E2 can not only increase the proliferation of granulosa cells(GCs),but also suppress GC apoptosis and follicular atresia.Our previous study showed that CYP19A1 gene was down-regulated during porcine follicular atresia,but the mechanism of this down-regulation and its role in regulating follicular atresia remain unknown.In this study,we focus on CYP19A1 gene in porcine granulose cells(pGCs)to analyze its role in regulating pGC apoptosis and E2 synthesis.The mechanisms of CYP19A1 regulation by SMAD4 and miRNAs are also explored.The results provide theoretical evidence to increase sow fertility.The results of this study are listing as follows:(1)CYP19A1 inhibits pGC apoptosis and increases E2 synthesisIn this study,through knockdown and over-expression of CYP19A1,we analyzed its role in regulating pGC apoptosis and E2 synthesis.Results show that knockdown CYP19A1 by small interference RNA(siRNA)dramatically reduced CYP19A1 expression level,significantly up-regulates pGC apoptosis and inhibits E2 concentration in cultural medium.Overexpression of CYP19A1 can significantly increase its expression level and inhibit pGC apoptosis,E2 secretion level is also significantly increased.The results confirm that CYP19A1 acts as an anti-apoptotic factor in pGCs and is essential for E2 secretion.(2)SMAD4 acts as transcriptional factor and directly regulates CYP19A1 expression in pGCsThe ovarian specific promoter of porcine CYP19A1 gene is identified(-785/-681)by dual-luciferase gene reporter system and two classical SMAD4 binding sites are found.Luciferase activity assay results suggest that SMAD4 can increase the activity of CYP19A1 promoter,ChIP assay is performed and proved that SMAD4 directly bind to SBE on the promoter of CYP19A1.FACS results show that SMAD4 inhibits pGC apoptosis through CYP19A1 and RIA results show that SMAD4 increases E2 secretion through CYP19A1.These results fully prove that SMAD4 acts as transcriptional factor and increases CYP19A1 gene transcription by directly binding to CYP19A1 promoter,therefore increases E2 synthesis and inhibits pGC apoptosis.(3)SMAD4 regulates CYP19A1 expression through miR-126*/FSHR axisBy using bioinformatic analysis and relative molecular technology,we prove that FSHR is a functional targets of miR-126*,which dramatically inhibits FSHR expression and increases pGC apoptosis.Two SBE are found in the promoter of miR-126*through sequence analysis.Luciferase activity assay is performed and prove that SMAD4 can bind to miR-126*promoter and inhibit miR-126 expression level,further increases FSHR expression level.Thus,qRT-PCR and western blot assay proved that SMAD4 increases CYP19A1 expression through miR-126*/FSHR axis.Results above show that SMAD4 acts as transcription factor and indirectly regulates CYP19A1 expression through targeting miR-126*/FSHR axis.(4)miRNAs regulates CYP19A1 expression at post-transcription levelThrough biology informatic analysis and follicular atresia miRNA array,we find that CYP19A1 is a potential target of miR-lOb and miR-146b-5p.In pGC cultured in vitro,we prove that both miR-10b and miR-146b-5p can bind to CYP19A1 3'-UTR and inhibit its expression through luciferase activity assay,qRT-PCR and western blot.FACS assay confirms that miR-10b and miR-146b-5p act as apoptotic factors and increase pGC apoptosis.RIA results show that miR-10b inhibits E2 secretion through targeting CYP19A1.Our results prove that miR-10b and miR-146b-5p can regulate CYP19A1 expression and functions at post-transcription level.In summary,we prove that CYP19A1 is essential for maintaining pGCs state and E2 secretion.In pGCs,transcription factors,epigenetics factors and regulation axis(SMAD4,miR-10b,miR-146b-5p and miR-126*/FSHR axis)regulate CYP19A1 expression at transcription and post-transcription levels in direct or indirect ways.Research results reveal the functions of CYP19A1 and its expression regulation mechanisms in pGCs and provide theoretical foundation for further studies of porcine follilcular development and atresia.