The Influencing Mechanism Of MiRNA-148a-3p On The Function Of Goat Ovarian Granulosa Cells

miRNAs are highly expressed in the ovary and play an important role in regulating ovarian function.At present,many studies have reported that mi R-148a-3p plays an important role in disease and regulating cell proliferation and apoptosis,but there are few reports on its effect on the function of goat ovarian granulosa cells.In this study,ovarian granulosa cells of Dazu black goat were used as experimental objects,and by overexpression and inhibition of miRNA-148a-3p,the the effects of miRNA-148a-3p on granulosa cell function were investigated by q RT-PCR,Western Blot,flow cytometry,ELISA and other techniques and methods,which lays the foundation for further understanding of the mechanism of miRNA-148a-3p regulating follicle growth and development.The results are as follows:1.After overexpression or inhibition of miRNA-148a-3p,CCK8 was used to detect the proliferation activity of goat ovarian granulosa cells after transfection for different time.The results showed that the proliferation activity of the overexpression group was significantly higher than that of the control group for 72 h of transfection(P<0.01),and the cell proliferation activity in the inhibition group was also significantly lower than that in the control group at 72 h after transfection(P<0.05).The Ed U detection result showed that the proportion of Ed U positive cells in the overexpression group was obviously higher than that in the control group(P<0.01).Further analysis by q RT-PCR found that expression of PCNA at the m RNA and protein levels in the miRNA-148a-3p overexpression group were significantly increased compared with the control group(P<0.01),while significantly decreased compared with the control group in the inhibition group(P<0.05).2.By flow cytometry,the apoptosis of granulosa cells after overexpression or inhibition of mi R-148a-3p was detected.The results found that,the apoptosis rate of miRNA-148a-3p was significantly lower than that of the control group in the overexpression group(P<0.01),and significantly higher than that of the control group in the inhibition group(P<0.05),indicating that overexpression of miRNA-148a-3p reduced the apoptosis rate of granulosa cells.Further detection found that the BCL2/BAX ratio in the miRNA-148a-3p overexpression group was significantly higher than that in the control group(P<0.01),while there was no obvious difference in the BCL2/BAX ratio between the miRNA-148a-3p inhibition group and the control group(P>0.05).The results indicated that mi R-148a-3p could inhibit apoptosis of ovarian granulosa cells.3.After overexpression or inhibition of miRNA-148a-3p for 24 h,the autophagy of granular cells was detected by transfection adenovirus.The results showed that overexpression of miRNA-148a-3p significantly promoted the autophagy of granular cells,while inhibition of miRNA-148a-3p had no significant change.Further westernblot analysis found that overexpression of miRNA-148a-3p significantly up-regulated LC3-II /LC3-I ratio(P<0.05)and had no significant effect on p62 expression(P>0.05)and inhibition of miRNA-148a-3p significantly up-regulated the expression of p62(P<0.05)while had no significant effect on LC3-II /LC3-I ratio(P>0.05),which suggested that overexpression of miRNA-148a-3p promoted autophagy in granulose cells.4.The content of steroid hormones in the culture medium of granulosa cells transfected with miRNA-148a-3p for 72 h were detected by ELISA kit.It was found that the progesterone content in miRNA-148a-3p overexpression group was significantly higher than that in NC group(P<0.01),but the estradiol content had no significant change(P>0.05).The content of progesterone in cell culture medium in miRNA-148a-3p inhibition group was significantly lower than that in control group(P<0.01),while the content of estradiol had no significant change(P>0.05),which indicated that miRNA-148a-3p promoted progesterone secretion but had no effect on estradiol secretion in granulosa cellsThe above results suggested that miRNA-148a-3p promoted cell proliferation,inhibited apoptosis,induced autophagy and promoted progesterone synthesis in ovarian granulosa cells,which provided a theoretical basis for revealing the mechanism of miRNA-148a-3p regulating follicular development and atresia.