| The proliferation and differentiation of granulosa cells are critical for ovarian follicular development, dysfunction of which may lead to ovarian disorders. Recently several studies identified that microRNAs are involved in regulating granulosa cell functions. In this study, we found that miR-181a expression was decreased during the growth of mouse ovaries and ovarian follicles, suggesting that miR-181a may play an important role in ovarian follicle development. However, the mechanism is largely unknown.The results of Quantitative real-time PCR (Q-PCR) and Cell Counting Kit-8 assay confirmed that miR-181a suppressed mouse granulosa cells (mGCs) proliferation. Overexpression of miR-181a in mouse granulosa cells decreased cyclin D2 and proliferating cell nuclear antigen (PCNA) expression in a dose-dependent manner, while cyclin D2 mRNA and protein level were significantly increased in mGCs after transfected with miR-181a inhibitor (50nM).The results of luciferase assay confirmed that miR-181a inhibited luciferase activity through binding to acvr2a 3’UTR. Furthermore, our results shown that the mRNA and protein level of activin receptor 2a was apparently reduced in mGCs infected with Ad-miR-181a and induced by miR-181a inhibitor performed by Quantitative real-time PCR (Q-PCR) and Western Blot. Otherwise, the level of acvr2a increased gradually in 3,8,12, and 21-day-old mouse ovaries and growing follicles, having an inverse relationship with miR-181a.Q-PCR results shown that activin A reduced miR-181a expression in dose and time-dependent manner. We found that miR-181a suppressed the promoting effect of activin A on mGCs proliferation, and also decreased activin A-induced cyclin D2 expression. Western Blot results shown that activin A enhanced Smad2 phosphorylation, and overexpression of miR-181a reduced the phosphorylation of Smad2 (Ser465/467). Meanwhile, miR-181a also inhibited the promoting effect of activin A on Smad2 phosphorylation. Moreover, activin A-induced genes expression, such as CYP19A1, P450scc, and ESR1, were dramatically decreased by miR-181a. Importantly, the result of Q-PCR demonstrated that miR-181a expression level was significantly increased in blood of premature ovarian failure patients.In summary, we find a significant increase in circulating levels of miR-181a in POF patients, and our data demonstrate that miR-181a suppresses mouse granulosa cells proliferation by targeting of activin receptor 2a. All of our studies suggest that miR-181a is an important modulator of ovarian follicular development, and aberrant expression of miR-181a in granulosa cells and ovaries may be invovled in the mechanism of premature ovarian failure. |
