The Expression Of MiRNA-130a In Mouse Follicles And Effects On Proliferation And Apoptosis Of Granulosa Cells

MicroRNA is an intracellular endogenous non-coding RNA molecules,whose Length is about22-24nt,it plays its major role in the negative regulation on gene expression and inhibit translation of a particular gene or its mRNA degradation mediated,thus participate in the regulation of cell proliferation differentiation and apoptosis processes.With the deepening of microRNA research in recent years, we also found that microRNA and its signaling pathway play an important role in the regulation of follicular development by using gene knockout or conditional knockout mouse model in addition. MicroRNA,in mouse ovaries,directly or indirectly, regulate expression of the target gene or mRNA in follicular development, thus participate in the process of follicular development in mice. The mouse are experimental subjects.Mir-130a expression levels will be detected in mouse at different developmental stages of follicles and follicular granulosa cells by real time PCR, to reveal the expression of mir-130a in mouse follicles and speculated its function. Then using the model of ovarian granulosa cells cultured in vitro, transfecteing granulosa cells with mir-130a mimics is to study granulosa cell proliferation and apoptosis further.The experimental results are as follows:(1) The expression of mir-130a in mouse follicles at different developmental stages has differences. we extracte miRNA from12d,21d,21d PMSG48h,21d PMSG48h hCG6h follicles, expression level of mir-130a will be detected by Q-PCR.We find that the expression of mir-130a is the highest level in small preantral follicles (100～130μm),then the expression level of mir-130a is significantly downregulated continuously in big preantral follicles (200～280μm), small antral follicles (450～550μm) and preovulatory follicles (500～600μm)(2) The expression of mir-130a in mouse granulosa cells at different developmental stages has differences.The results showed by real time PCR that the expression level of mir-130a in granulosa cells of big preantral follicles (200～280μm) was significantly downregulated, compared with that in granulosa cells of small preantral follicles(100～130μm)(P<0.05), and downregulated in granulosa cells of small antral follicles (450μm-550μm)(P<0.05), while significantly upregulated in granulosa cells of preovulatory follicles (500μm-600μm)(P<0.05). Compared with the expression pattern in different stages of follicles, the expression pattern of mir-130a in granulosa cells is not always identical.(3) mir-130a had no significant effect on cell proliferation in granulosa cells. We get mature follicle granulosa cells from newborn mice ovary21days after injecting PMSG48h and hCG6h. Mature follicular granulosa cells were isolated cultured and then transfected with mir-130a mimics,48h later, the granulosa cells were analyzed the proliferation rate by Microplate Reader. The results showed that mir-130a mimics had no significant effect on granulosa cell proliferation. It suggests that mir-130a may not participate in the process of granulosa cell proliferation.(4) mir-130a can significantly inhibit granulosa cell apoptosis. We get mature follicle granulosa cells from newborn mice ovary21days after injecting PMSG48h andhCG6h. The primary cultured granulosa cells were directly transfected with mir-130a mimics,48h later, the granulosa cells were analyzed the apoptosis rate by Flow Cytometer. The results showed that mir-130a mimics could significantly inhibit granulosa cell apoptosis. It suggests that mir-130a can inhibit granulosa cell apoptosis.