The Expression Of Mir-381in Different Stages Of Follicles And Its Roles In Granulosa Cells In Mice

MicroRNAs are a kind of endogenous small non-coding single-stranded RNA. In the human genome, more than60%of the genes will be affected by the regulation of microRNAs. Studies have shown that microRNAs can regulate cell proliferation, differentiation, apoptosis, early development of embryo, etc.In this research, mice were chosen as an experimental animal model, based on the expression pattern of mir-381in ovaries of different ages of mice or with super-ovulation hormones treatment, real-time PCR analysis was employed to study the expression pattern of mir-381in different stages of follicles and follicular granulosa cells. Furthermore, we studied the function of mir-381in granulosa cell proliferation, cell cycle and cell apoptosis by transfecting mir-381mimics or inhibitors and then we detected the influence of mir-381mimics or inhibitors on the expression of its potential target genes in cultured granulosa cells. The experimental results were described as follows:(1) The expression level of mir-381showed significant difference in different stages of follicles. The results showed that the expression level of mir-381in big preantral follicles (150μm-250μm) was significantly downregulated, compared with that in small preantral follicles (90μm-130μm)(P<0.05), while significantly upregulated in small antral follicles (450μm-550μm)(P<0.05), and downregulated in preovulatory follicles (500μm-600μm)(P<0.05).(2) The expression level of mir-381was also significantly regulated in granulosa cells of different stages. The results showed that mir-381was highly expressed in granulosa cells of small preantral follicles (90μm-130μm), and significantly downregulated in granulosa cells of big preantral follicles (150μm-250μm) and small antral follicles (450μm-550μm)(P<0.05), while significantly upregulated in preovulatory follicular granulosa cells (500μm-600μm)(P<0.05). Compared with the expression pattern in different stages of follicles, mir-381in granulosa cells was not always consistent, it could be implied that the expression level of mir-381in different stages of follicular granulosa cells was related to the differentiation of follicular granulosa cells, such as the formation of cumulus cells around oocytes, or the formation of antrums formation.(3) mir-381can significantly inhibit cell proliferation. Preantral follicular granulosa cells were isolated, cultured and then transfected with mir-381mimics or inhibitors,48h later, the proliferation rate of granulosa cells was analyzed. The results showed that mir-381mimics can significantly induce, but the inhibitors could significantly decrease, the expression of mir-381in granulosa cells. Furthermore, mir-381mimics can inhibit cell proliferation while mir-381inhibitors can promote cell proliferation. These results suggested that mir-381maybe has an inhibitory effect in the process of follicular granulosa cell proliferation. It was consistent with the result of the decline of mir-381as follicles growed.(4) mir-381affected cell cycle of granulosa cells. After transfection with mir-381mimics, the cell cycle of granulosa cells was analyzed. The results showed that over-expression of mir-381by its mimics can significantly upregulate the G1phase level. It suggested that mir-381can enable granulosa cells block in the G1/S phase, thus inhibiting the process of granulosa cell proliferation.(5) mir-381had no significant effect on cell apoptosis in granulosa cells. After transfection with mir-381mimics, the cell apoptosis of granulosa cells was analyzed. The results showed that over-expression of mir-381mimics had no significant effect on cell apoptosis. It suggested that mir-381may not participate in the process of granulosa cell apoptosis.(6) The preliminary study of target genes of mir-381in granulosa cells. In order to further reveal the regulation mechanism of mir-381in granulosa cell proliferation and cell cycle, we first predicted the target genes of mir-381by microRNAs target genes prediction softwares (TargetScan, DIANA-microT and PicTar). We preliminarily screened acvrl and acvr2a as the target genes related to cell proliferation and cdk2ap2, cdk-4, and cdk-6as the targets genes related to cell cycle, and then we further examined the effect of over-expression or down-regulation of mir-381on the expression level of these potential target genes by transfecting mimics/inhibitors. The results showed that the expression level of acvrl, acvr2a, cdk2ap2, cdk-4and cdk-6were significantly downregulated by mir-381mimics and upregulated by mir-381inhibitors. It suggested that these genes may be the targtet genes of mir-381. Thus we presumed that mir-381may regulate granulosa cell proliferation through acvrl/acvr2a and regulate the cell cycle through cdk2ap2/cdk-4/cdk-6.In conclusion, this study revealed the expression pattern of mir-381in different stages of follicles and the corresponding granulosa cells in mice, analyzed the regulatory function of mir-381in granulosa cell proliferation and cell cycle. Besides we preliminarily identified the target genes of mir-381in granulosa cells. The research provided an important information for further understanding of the regulatory mechanism of follicular develonment and granulosa cell proliferation.