microRNA-130a抑制KGN细胞FOXL2基因的表达及对细胞增殖的影响

Inhibition of FOXL2 gene expression and affection of proliferation in KGN cells by target microRNA-130a

目的:研究外源性microRNA-130a在卵巢颗粒细胞KGN中过表达对FOXL2基因表达的影响,探索microRNA-130a对KGN细胞增殖的影响。方法:人工设计合成靶向FOXL2基因的microRNA-130a模拟物,脂质体法将microRNA-130a模拟物转染入KGN细胞中,Trizol法和RIPA蛋白裂解法分别获取高纯度的细胞总RNA和总蛋白,实时荧光定量聚合酶联反应(FQ-PCR)检测microRNA模拟物转染后细胞中FOXL2基因mRNA表达水平,Western Blot检测FOXL2蛋白表达水平,四甲基偶氮唑盐法(MTT)检测microRNA-130a转染后KGN细胞的增殖情况。结果:成功转染KGN细胞后,阴性对照组FOXL2基因mRNA和蛋白的表达水平与空白对照组相比无明显的差异(P>0.05),mi R-130a转染组能显著抑制FOXL2 mRNA和蛋白的表达,与空白对照组和阴性对照组相比均有统计学差异(P<0.05)。MTT法检测各组KGN细胞的OD值及细胞增值率,microRNA-130a转染后能明显促进KGN细胞的增殖,与与空白对照组和阴性对照组相比均有统计学差异(P<0.05)。结论:外源性的mi R-130a对FOXL2基因mRNA和蛋白表达有明显的抑制作用,且能明显促进KGN细胞的增殖。

Objective To study the regulative effect on FOXL2 expression and cell proliferation of exogenous microRNA-130 a overexpression in ovarian granulosa cells. Methods The microRNA-130 a mimics were artificially synthesized and transfected in KGN cells by using Lipofectamine 3000 Reagent. High-purity cellular total RNA and total proteins were obtained by Trizol and RIPAlysis methods respectively. The FOXL2 mRNA were detected by real-time fluorescent quantitative polymerase chain reaction（FQ-PCR）, and FOXL2 protein expression levels were detected by Western blot. MTT assay was used to determine the effect of target microRNA-130 a on KGN cells proliferation. Results After the transfection of miR-130 a mimics in KGN cells, the expressions of FOXL2 mRNA and FOXL2 protein were significantly down-regulated compared with the blank control group and negative control group（P 0.05）, and the expressions of FOXL2 mRNA and FOXL2 protein in negative control group compared with the blank control group had no significant difference（P0.05）. The OD value and cell proliferation rate were determined by MTT assay, the miR-130 a can obviously promote KGN cell proliferation compared with the blank control group and negative control group（P 0.05）. Conclusion Exogenous miR-130 a can significantly inhibit the expression of FOXL2 gene,and promote the proliferation of KGN cells.