The Mechanism Of MiR-24-3p Regulating Estradiol Synthesis,proliferation And Apoptosis Of Porcine Ovarian Granulosa Cells

Reproduction is a key part in pig production,and the level of fecundity directly affects the economic benefits of pig production and the development of the pig industry.The level of follicle development plays an important role in the reproductive process of sows,and granulosa cells are the largest cell population in follicles,and their physiological functions such as estrogen synthesis,proliferation and apoptosis will affect follicular growth and atresia.Therefore,it is very important to explore the factors that affect the physiological function of granulosa cells.MicroRNA(miRNA)is a non-coding RNA of about 22 nucleotides in length,which mainly degrades or reduces the stability of mRNA by pairing with the 3’UTR of target gene mRNA,and studies have shown that miRNA is involved in regulating the physiological function of granulosa cells.miR-24-3p has been proved to be highly expressed in the ovaries of high-yield sows,and the expression of miR-24-3p in dominant follicles is significantly higher than that in ordinary follicles.Therefore,The porcine ovarian granulosa cells were taken as the research object to clarify the role and mechanism of miR-24-3p in granulosa cells.In this experiment,miR-24-3p mimics and inhibitor treatment were used to clarify the role of miR-24-3p in estradiol synthesis,proliferation and apoptosis of granulosa cells.The direct target genes of miR-24-3p in regulating granulosa cell function were identified by online software prediction and dual luciferase reporter assay.The results of this test are as follows:1.miR-24-3p promotes estradiol synthesis in granulosa cellsIn porcine ovarian granulosa cells,overexpression of miR-24-3p can significantly increase the concentration of estradiol,and significantly up-regulate the transcription and translation levels of St AR,CYP19A1 and CYP11A1(P<0.05).Interfering with miR-24-3p inhibited the synthesis of estradiol,and significantly down-regulated the mRNA levels of CYP11A1 and CYP19A1 and the protein levels of CYP11A1 and CYP19A1(P<0.05).2.miR-24-3p promotes the proliferation of granulosa cellsOverexpression of miR-24-3p promoted the proliferation of granulosa cells,significantly up-regulated the transcription and translation of CDK4,Cyclin B,Cyclin D,and significantly up-regulated the mRNA level of Cyclin E(P<0.05).Interfering with miR-24-3p inhibited the proliferation of granulosa cells,significantly down-regulated the expressions of CDK4,Cyclin B and Cyclin D,and down-regulated the protein level of Cyclin E(P<0.05).3.miR-24-3p inhibits apoptosis of granulosa cellsOverexpression of miR-24-3p significantly reduced the ratio of early apoptotic and late apoptotic cells,significantly down-regulated the mRNA and protein levels of BAX and up-regulated the level of BCL-2 mRNA,and the ratio of BCL-2/BAX was significantly increased at the protein level(P<0.05).Inhibition of miR-24-3p significantly up-regulated the proportion of early apoptotic cells,down-regulated the mRNA level of BCL-2 and up-regulated the mRNA and protein levels of BAX.At the same time,the ratio of BCL-2/BAX was significantly down-regulated at the protein level(P<0.05).4.miR-24-3p targets P27 to regulate the proliferation and apoptosis of granulosa cells,and regulates estradiol synthesis in granulosa cells by targeting TOP1Through online software analysis and dual-luciferase reporter experiments,it was found that both P27 and TOP1 are the direct target genes of miR-24-3p.Overexpression of miR-24-3p inhibited the transcription and translation of P27 and TOP1,while interfering with miR-24-3p promoted the expression of P27 and TOP1(P<0.05).Overexpression of P27 could attenuate the promoting effect of miR-24-3p on the proliferation of granulosa cells and the inhibitory effect on apoptosis,while overexpression of TOP1 could attenuate the promoting effect of miR-24-3p on estradiol synthesis in granulosa cells.In conclusion,miR-24-3p promotes the proliferation of granulosa cells and inhibits their apoptosis by targeting P27.And miR-24-3p can also promote estradiol synthesis in granulosa cells by targeting TOP1.