| Physiological regulation of the ovaries involves a variety of cytokines and signaling pathways. Ovarian granulosa cells, whose proliferation, differentiation and apoptosis are related to the development, maturation and even atresia of the follicle.microRNAs are the small non-coding RNAs, which was involved in the regulation of many physiological functions, but the function study in the process of pig ovarian follicular development is less. Therefore, we used previous microRNAs deep sequencing results in the mice ovaries, with the preliminary research have been done in mice follicles, we aimed select to that three kinds of microRNAs, which were miR-130a/16/381, had a significant influence on the development of mouse follicles and the growth and functions of the granulosa cells, to further detect the expression of these three microRNAs in granulose cells, and studied the function roles of miR-130a/16/381 in porcine ovarian granulose cells, including the proliferation, differentiation, apoptosis and the secretion of hormones. The results are as follows:(1) RT-PCR was used to detect the endogenous expressions of miR-130a, miR-16 and miR-381 in porcine granulosa cells. The results showed that the three miRNAs were expressed in porcine granulosa cells.(2) Effects of microRNAs overexpression on cell cycle, proliferation and apoptosis of porcine ovarian granulosa cells. The results showed that after these three miRNAs were over expressed, which had no significant effect on the apoptosis of granulosa cells. Over expression of miR-130a could significantly promote cell proliferation (P<0.001), and reduce the number of G1 phase cells (P<0.05) and keep the majority of cells arrested in S phase (P<0.01). Over expression of miR-16 could significantly inhibit cell proliferation (P<0.001), with a significant reduction of PCNA mRNA level (P<0.05), and significantly decrease the number of G2 phase cells (P<0.01). Over expression of miR-381 could significantly inhibit cell proliferation (P<0.01), with a significant reduction of PCNA mRNA level (P<0.01), and have no significant effect on cell cycle.(3) Effect of microRNAs overexpression to the estrogen and progesterone secretion in porcine ovarian granulosa cells. The results showed that over-expression of these three microRNAs respectively all could significantly promote the secretion of estrogen and progesterone in granulosa cells (P<0.01). Real time-PCR detection of steroid hormone relevant genes showed that over-expression of miR-130a significantly reduced the expression of StAR (P<0.001) and CYP11A1 (P<0.05), increased the expression of CYP19A1 (P<0.05), while had no significant effect on HSD3β mRNA level; over-expression of miR-16 significantly reduced the expression of StAR (P<0.001) and CYP11A1 (P<0.05) and had no significant effect on the mRNA levels of CYP19A1 and HSD3β; over-expression of miR-381 could significantly decrease the expression of StAR (P<0.001), increase the expression of HSD3β (P<0.05) and have no significant effect on the expression of CYP11A1 and CYP19A1.The results show that miR-130a/16/381 influence porcine ovarian granulose cells growth and the function of hormone secretion. The results laid a foundation for further research on the functions of these three kinds of microRNAs in the development and maturation of porcine follicle. |
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