Study Of Regulation About MiR-101 And MiR-144 On Ovarian Granulosa Cells In Dairy Goat

Mi RNAs are widely expressed in mammalian ovaries and paly regulatory roles in follicular growth and development and ovarian function.Mi R-101 and miR-144 regulate the growth,differentiation and apoptosis of various cells,but there is no study of miR-101 and miR-144 regulation of Guanzhong dairy goat ovarian granulosa cells.In the present study,the target genes of miR-101 and miR-144 were verified by bioinformatics and dual luciferase vector.The effect of miR-101 and miR-144 on the target gene TGIF1 was studied by RT-q PCR and Western blot.The mechanism of miR-101 and miR-144 on ovarian granulosa cell apoptosis was studied by flow cytometry.The aim of this study was to elucidate the mechanism of miR-101 and miR-144 in regulating ovarian granulosa cells,and to provide experimental evidence for improving the lambing rate of dairy goats.The main findings are as follows:1.To investigate the relationship between miR-101 and miR-144 and TGIF1 gene in ovarian granulosa cells of dairy goat.(1)Using the bioinformatics software to predict the 3'UTR region of the TGIF1 gene containing the target site of miR-101 and miR-144.Mi R-101 and miR-144 mimics and Negative control(NC)and TGIF1-3'UTR double luciferase reporter vector were co-transfected into 293 T cells and detected luciferase activity.The results showed that miR-101 and miR-144 significantly inhibited the activity of luciferase compared with NC group.The miR-101 and miR-144 mimics and NC were co-transfected with mutant vector to detect luciferase activity.The results showed that miR-101 and miR-144 had no significant effect on the luciferase activity of the mutant vector compared with the NC group.The TGIF1 gene was preliminarily identified as the target gene of miR-101 and miR-144.(2)The expression of TGIF1 gene in miR-101 and miR-144 was detected by RT-q PCR and Western blot.The expression of TGIF1 gene in goat was also detected.Mimetic and inhibitors of NC,miR-101 and miR-144 were transfected into dairy goat ovarian granulosa cells.The results showed that miR-101 and miR-144 significantly inhibited the expression of TGIF1 m RNA and protein compared with NC group.After transfection of miR-101 and miR-144 inhibitors,the expression of the TGIF1 gene was significantly up-regulated at m RNA and protein levels.At the same time,according to the results of RT-q PCR,the expression of TGIF1 gene was relatively high in the uterus,ovary and mammary gland of dairy goat.2.To study the regulatory mechanism of miR-101 and miR-144 on ovarian granulosa cell apoptosis in dairy goat.(1)The effect of miR-101 and miR-144 on the apoptosis of ovarian granulosa cells in dairy goat was detected by flow cytometry.The results showed that miR-101 and miR-144 significantly increased the apoptotic rate of granulosa cells compared with NC group.(2)Western blot was used to detect the expression such as Bcl-2,Bax and p53.The results showed that miR-101 and miR-144 significantly down-regulated the protein level of Bcl-2 and Bax apoptotic genes and the ratio of Bcl-2 / Bax was decreased.Compared with NC group,miR-101 and miR-144 significantly up-regulated the phosphorylation level of p53.The results showed that the expression of TGIF1 gene in the uterus,ovary and mammary gland of dairy goat is relatively high.Mi R-101 and miR-144 negatively regulated the expression of target gene TGIF1 in dairy goat ovarian granulosa cells.Mi R-101 and miR-144 may promote the apoptosis of dairy goat ovarian granulosa cells by down-regulating the ratio of Bcl-2 / Bax and up-regulating the phosphorylation level of p53.