miR-383通过下调细胞周期相关蛋白的表达抑制小鼠卵泡颗粒细胞的增殖

miR-383 inhibits proliferation of granulosa cells by down-regulation of cell cycle-related proteins in mice

目的探讨微小RNA-383(miR-383)在小鼠卵泡不同发育阶段的表达规律以及对颗粒细胞增殖的影响。方法取健康雌性受孕昆明小鼠不同小鼠出生后12、21、21 d[腹腔注射10 IU孕马血清促性腺激素(PMSG)48 h后]和21 d[腹腔注射10 IU PMSG 48 h和10 IU人绒毛膜促性腺激素(hCG)6 h后]的卵巢组织、卵泡和颗粒细胞,通过实时定量PCR法检测miR-383的表达情况。取小鼠出生21 d的颗粒细胞,分别转染miR-383模拟物(miR-383 mimics)或miR-383抑制物(miR-383 inhibitor),另外设置阴性对照组。通过噻唑蓝(MTT)法检测颗粒细胞增殖情况,流式细胞术检测颗粒细胞的细胞周期变化。采用Western blot法检测细胞周期相关蛋白细胞周期蛋白D1(cyclin D1)、cyclin B、细胞周期蛋白依赖激酶1(CDK1)、CDK2、CDK4的蛋白水平。结果与小腔前卵泡相比,大腔前卵泡中miR-383的含量降低,但是在腔较小的有腔卵泡中含量升高,成熟卵泡中的含量又进一步降低。与小腔前卵泡颗粒细胞相比,大腔前卵泡和腔较小的有腔卵泡颗粒细胞中miR-383的含量降低,但是在成熟卵泡颗粒细胞中的含量却有所上调。与空白组相比,miR-383 mimics组颗粒细胞的增殖速度减慢,且G0/G1期比例升高,G2/M期比例降低,cyclin D1、cyclin B、CDK1、CDK2、CDK4蛋白水平降低;而miR-383 inhibitor组颗粒细胞的增殖速度加快,G0/G1期比例降低,G2/M期比例升高,且cyclin D1、cyclin B、CDK1、CDK2、CDK4蛋白水平增加。结论miR-383可通过抑制细胞周期相关蛋白的表达,将颗粒细胞周期阻滞在G0/G1期,进而抑制卵泡颗粒细胞的增殖。

Objective To investigate the expression of microRNA-383(miR-383)in viability classification of growing follicles and its biological function on proliferation and cycle of granulosa cells.Methods The expression of miR-383 in ovaries tissues,follicles and granulosa cells extracted from Kunming mice after birth of 12,21 days,21 days[after injection of pregnant mare serum gonadotropin(PMSG)for 48 hours]and 21 days[after injection of PMSG for 48 hours and(human chorionic gonadotrophin)hCG for 6 hours]were detected by real-time quantitative PCR.The miR-383 mimics or miR-383 inhibitor were transfected into granulosa cells extracted from Kunming mice after birth of 21 days,respectively as miR-383 mimics group and miR-383 inhibitor group.Meanwhile,we established negative control(NC)group.MTT assay was used to detect the proliferation and flow cytometry was used to detect cell cycle of granulosa cells.And the levels of cyclin D1,cyclin B,CDK1,CDK2,and CDK4 protein were tested by Western blot analysis.Results Compared with small preantral follicles,miR-383 level in big preantral follicles and big antral follicles were both lower,but miR-383 in small antral follicles was higher.Similarly,miR-383 of granulosa cells in big preantral follicles and small antral follicles were lower than that of granulosa cells in small preantral follicles,but up-regulated in granulosa cells of big antral follicles.Compared with granulosa cells in the NC group,the proliferation activity of granulosa cells in miR-383 mimics group was lower,and there was more granulosa cells in G0/G1 phase,less in G2/M phase in miR-383 mimics group,besides cyclin D1,cyclinB,CDK1,CDK2,CDK4 proteins in miR-383 mimics group were all lower.Meanwhile,the proliferation activity of granulosa cells in miR-383 inhibitor group was higher,and there was less granulosa cells in G0/G1 phase,more in G2/M phase in miR-383 inhibitor group,besides cyclinD1,cyclinB,CDK1,CDK2,CDK4 proteins in miR-383 inhibitor group were all higher than those proteins in the NC group.Conclusion miR-383 inhibits the proliferation of granulosa cells by down-regulating of cycle-related proteins and blocking granulosa cells in G0/G1 phase.