Regulatory Role Of MiRNA-375 In Expression Of BMP15/GDF9 Receptors And Its Effect On Proliferation And Apoptosis Of Bovine Cumulus Cells

Oocyte-derived GDF9 and BMP15 are important regulators of follicular growth and ovarian function.Through autocrine and paracrine mechanisms,these two factors can regulate cell differentiation,proliferation,and other functions in local ovary.Furthermore,GDF9 and BMP15 play vital roles in follicular growth,atresia,ovulation,fertilization,reproduction,and maintenance.Numerous studies have demonstrated a synergy between BMP15 and GDF9,exhibiting an additive effect.Research indicated the synergistic effect of BMP15 and GDF9 was mainly mediated via BMPR2,a target gene of miRNA-375,whose overexpression would induce the apoptosis in bovine cumulus cells.However,it is not clear which genes mediate and regulate the additive effect of the BMP15 and GDF9 on bovine cumulus cells(CCs).To investigate the effect and the regulatory mechanism of miRNA-375 on the proliferation and apoptosis of bovine cumulus cells,following experiments were conducted.Firstly,bovine cumulus cells were separately incubated in vitro,and miRNA-375 mimics/miRNA-375 inhibitor were applied to the overexpression and inhibition of miRNA-375 in these cells,respectively.Flow cytometry,fluorescent quantitation PCR and CCK-8 were utilized to detect the effect of miRNA-375 on proliferation and apoptosis in bovine cumulus cells.Results indicated miRNA-375 overexpression inhibited the in vitro proliferation of bovine cumulus cells,while the inhibition on miRNA-375 expression had no significant effect on in vitro proliferation.Meanwhile,overexpression of miRNA-375 resulted in a significantly elevated cell apoptosis rate(p<0.05),while the inhibition on miRNA-375 expression had no significant effect on cell apoptosis,show that the role of miRNA-375 on cumulus cell proliferation and apoptosis is through its target gene BMP15 function respectively or interaction with GDF9 on co-receptor BMPR2.To further clarify the mechanism of miRNA-375 in the proliferation and apoptosis of bovine cumulus cells,q PCR and Western Blot were used to detect changes of genes,namely ALK4,ALK5,ALK6,ALK7,BMPR2,Bax,Bcl-2,after miRNA-375 overexpression or inhibition.After miRNA-375 overexpression,no significant changes in ALK4,ALK5,ALK6 m RNA were found,with BMPR2,ALK7 and Bcl-2 expression significantly reduced,Bax expression significantly elevated;after miRNA-375 inhibition,no significant changes in ALK4,ALK5,ALK6,Bax,Bcl-2 were found,while the expressions of BMPR2 and ALK7 were elevated;in order to clarify whether the changes in genes were caused by miRNA-375 or its target gene BMPR2,we conducted BMPR2 knock-out in bovine cumulus cells.Results indicated the alteration in each gene after BMPR2 knock-out was in accordance with the changes of the genes in miRNA-375 mimics transfection group.These results suggest that miRNA-375 can regulate the expression of type I receptor ALK7 by specifically regulating the BMPR2 of BMP15 and GFD9 in the common receptors of many BMP15 and GDF9.Thus affecting the biological effects of BMP15 and GFD9 on the proliferation of cumulus cells.At the same time,miRNA-375 can also affect the expression of Bcl-2 and Bax to regulate the apoptosis of cumulus cells.This study provided certain theoretical evidence for clarifying influence factors in bovine cumulus cell proliferation and apoptosis,exploring bovine cumulus cell functions,and explaining the molecular mechanisms in follicular development,atresia and maturation.