| MicroRNAs are small, non-coding endogenous RNA, which regulate gene expression at post-transcriptional level and play important roles in many biological processes. In order to investigate the expression, function and regulation mechanism of microNRAs in the development of the follicles, microRNAs deep sequencing technology was employed in previous studies to explore the microRNAs profiling in mice after birth or with super-ovulation hormone treatment which was on behalf of the different stages of the follicles. In the present study we used real time PCR to further detect the expression profile of the miRNAs which was either differential expression or high expression (mir-871and mir-351) in different stages of follicles and corresponding granulosa cells was studied. Furthermore, we used in-vitro culture model of mice granulosa cells and preantral follicles to find the effects of microRNAs on proliferation and apoptosis of granulosa cells and follicular development. Meanwhile, the targets of microRNAs was predicted and analyzed by the methods combining bioinformatics with experimental validation. The results provided important information about the regulation mechanism of microRNAs involved in follicular development and granulosa cell function. The main findings are given as the following:1. The expression profile of mir-351and mir-871in different development stages of follicles was detected by Real-time PCR. The results showed that mir-351was highly expressed in the smaller preantral follicles (90-120μm), and significantly decreased in the secondary follicle (the bigger preantral follicles)(150-250μm)(P<0.01), but was significantly increased in the small antral follicles (450-550μm)(P<0.01), and then significantly decreased in the big antral follicles (500-600μm)(P<0.01). The expression of mir-871was also highly expressed in the smaller preantral follicles (100-130μm) and significantly decreased with the increasing of the diameter of follicles. The results indicated that mir-351and mir-871was differentially expressed during the development of follicles. We also detected the expression of mir-191, mir-470and mir-199a and found the similar expression pattern.2. The expression of miR-351and mir-871in granulosa cells of different development stages of follicles. The results showed these three microRNAs were all highly expressed in granulosa cells, with the similar expression pattern as in the corresponding follicles. 3. The effects of over-expression or knockdown of mir-351on granulosa cells proliferation and apoptosis. We found that overexpression of mir-351significantly induced the apoptosis of granulosa cells (P<0.01), and knockdown of mir-351can significantly suppress the apoptosis level of granulosa cells (P<0.01). This suggested that mir-351participated in the regulation of apoptosis of granulosa cells. We also found that mir-351has no effect on the proliferation of granulosa cells. Furthermore, the results indicated that mir-871has no effect on apoptosis and proliferation in mouse granulosa cells.4. Target prediction and studies of mir-351. Using software of TargetScanMouse, microRNA.org, miRWalk, miRNA Entry, and miRDBabase to predict the potential targets of mir-351, we found that IGF1R, EGF, Stat3, Mapkl4, and Bmf as the potential target of mir-351. To futher test the effect of mir-351on the expression of the potential target gene in granulosa cells, mir-351mimics or inhibitors was used in in vitro culture of granulosa cells. The results showed that the mRNA level of IGF1R, EGF, Stat3, and Mapk14was significantly decreased (P<0.01) by mir-351mimics, but was significantly increased by mir-351inhibitor (P<0.01). These results suggested that these potential target genes might be directly or indirectly regulated by mir-351in granulosa cells.5. mir-351was involved in the information of antral follicles. The preantral follicles were transfected with mir-351Mimics and Inhibitors,culturing in vitro,we found that the antral rate was respectively51.8%and25.7%in the group of mir-351Mimics NC and mir-351Mimics,and the antral rate was45.5%and57.6%in the group of mir-351Inhibitor NC and Inhibitors,this results indicated that as the developing of follicular development in vitro,mir-351Mimics could decreas the rate of antral follicles,and mir-351Inhibitor could increase it. This result indicated that mir-351could inhibit the information of antral follicles through inducing the apoptosis of granulosa cells.In concolusion, this study revealed the expression pattern of mir-351, mir-871, mir-191, mir-470and mir199a in different developmental stages of follicles and corresponding granulosa cells, and found out that mir-351was involved in the regulation of granulosa cell apoptosis. The potential target genes of mir-351was also tested in granulosa cells. The results of this study could provide important information for further understanding of the regulation of follicular development and follicular atresia. |
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