miR-let-7b对卵泡颗粒细胞发育的作用

MiR-let-7b Attenuates Proliferation Activities and Development of Follicular GranulosaCells via Targeting MAP3K1 Gene

目的:目前为止,尚不清楚micro-RNA(miR)-let-7b是否通过MAP3K1调节卵泡颗粒细胞(FGCs)凋亡,本研究拟运用PCR和Western blotting方法对此进行实验验证.方法:应用CCK-8测定细胞存活率.设计合成miR-let-7b和MAP3K1 mRNA特异性引物,利用qRT-PCR和Western Blotting分析FGCs中miR-let-7b和MAP3K1 mRNA基因与蛋白的表达水平.结果:随着miR-let-7b模拟物剂量(40、60、80、100、120μM)的增加,FGC的增殖活性逐渐降低,MIM-4组的增殖活性明显低于CG和MIM-1组(P<0.05).miR-let-7b表达水平随着模拟物剂量的增加而逐渐降低,MIM-3和MIM-4组的miR-let-7b水平低于MIM-1(P<0.05或P<0.01).MIM-3和MIM-4组MAP3K1 mRNA和蛋白水平显著低于CG,而且MIM-4组MAP3K1 mRNA和蛋白水平低于MIM-1组(P<0.05).结论:miR-let-7b可明显降低FGC增殖活性.下调FGC中miR-let-7b mRNA表达水平,miR-let-7b处理可剂量依赖性地降低MAP3K1 mRNA和蛋白质表达水平.

Objective Up to date,it has undetermined if the apoptosis of follicular granulosa cells(FGCs)is mediated by miR-let-7b via MAP3K1.Methods FGCs were transfected with miR-let-7b mimic at thedifferent doses(40,60,80,100 and 120μM),respectively.Expression levels of miR-let-7b and MAP3K1 mRNAs and proteins were determined using RT-PCR and Western blots.Luciferase report assay was applied to verify the targeting relationship between miR-let-7b and MAP3K1.Results The proliferation activity of FGCs in MIM-4 group was significantly less than those of CG and MIM-1 group(P<0.05).MiR-let-7b mimic obviously reduced expression levels of miR-let-7b in FGCs.The largest reduction was found in MIM-4.Levels of MAP3K1 mRNAs and proteins in MIM-3 and MIM-4 groups were lower than that of CG(P<0.05 or P<0.01).Co-transfection of miR-let-7b mimic inhibited significantly luciferase activity(P<0.05)as compared with GC.Conclusion The miR-let-7b could obviously depress the cell viability and accelerate apoptosis of ovine FGCs.Higher doses of miR-let-7b mimic(80μM and 100μM)could significantly depress expressions of miR-let-7b mRNAs,MAP3K1 mRNAs and proteinsin theovine FGCs.MiR-let-7b promoted the apoptosis of FGCs by inhibiting the MAP3K1 gene.