MiR-145靶向作用激活素受体ⅠB抑制小鼠卵巢颗粒细胞增殖

MiR-145 targeting activator receptor IB inhibits ovarian granulocyte proliferation in mice

目的探讨miR-145对小鼠卵巢颗粒细胞增殖的调控作用。方法制备Ad-miR-145重组腺病毒,分离培养3周龄的SPF级ICR雌性小鼠卵巢颗粒细胞(mGC),以不同浓度的Ad-miR-145(0 mol、50 mol、100 mol)感染m GC,48 h后通过CCK-8增殖实验检测细胞的增殖活力;收集细胞通过TRIZOL法提取细胞总RNA,反转录后进行实时定量PCR检测细胞增殖相关基因细胞周期蛋白cyclin D2 mRNA的表达情况;提取细胞蛋白,通过Western blot检测其中cyclin D2的蛋白水平;同时采用双荧光素酶报告基因调查miR-145对其预测靶基因激活素受体ⅠB(ACVRⅠB)的调控作用。结果成功获得滴度为8.91×10^(10) ifu/mL的高表达miR-145腺病毒,该腺病毒可在mGC中高表达miR-145;腺病毒介导的miR-145高表达可抑制m GC的增殖活力,并可抑制m GC中cyclin D2的m RNA和蛋白的表达;miR-145可抑制ACVRⅠB 3’-UTR的荧光素酶活性。结论 miR-145通过靶向作用ACVRⅠB抑制小鼠m GC增殖。

Objective To investigate the effect ofmiR-145 on the proliferation of ovarian granulosa cells in mice. Methods Adenoviruses harboring microRNA-145 mRNA （Ad-miR-145） were packaged, amplified and purified. Mouse granulosa cells （mGCs）, which were collected from ovaries of 21-day-old immature ICR mice through follicle puncture, were cultured normally and treated with Ad-mig-145 in different concentrations （0 mol, 50 mol, 100 mol）. The proliferation activity of the cells was detected by CCK-8 proliferation assay after 48 h. The total RNA was extracted by TRIZOL method and the expression of cyclin D2 mRNA was detected by real-time quantitative PCR. The expression of miR-145 was predicted by double luciferase reporter gene in predicting target gene activin receptor IB （ACVR I B）. Results The miR-145 adenovirus with high titer of 8.91 × 1010 ifu/ml was highly expressed in mGC, and the miR-145 mediated by miR-145 could inhibit the proliferation ofmGC, and suppressed the mRNA and protein expressions of cyclin D2 in mGCs. miR-145 could inhibit luciferase activity ofACVR I B Y-UTR. Conclusions MiR-145 can suppress mouse granulosa cells proliferation by targeting ACVR I B.