miR-133b靶向调控FOXL2对COV434细胞迁移增殖的影响

Effect of miR-133b targeted regulation of FOXL2 on migration and proliferation of COV434 cells

目的建立miR-133b在人卵巢颗粒肿瘤细胞(COV434)中的过表达模型,探讨其对FOXL2基因表达的影响,及其对COV434细胞的迁移增殖的作用。方法以生物信息学方法分析miR-133b是否靶向作用于FOXL2基因的UTR区域,依据预测结果设计合成靶向FOXL23′UTR的miR-133b mimics,运用脂质体法将miR-133b mimics瞬时转染入COV434细胞中,观察荧光以确定转染时间;miR-133b mimics转染人卵巢颗粒肿瘤细胞后,使用qRT-PCR检测COV434细胞中FOXL2基因mRNA的表达水平,使用Western Blot法检测FOXL2蛋白表达水平,细胞划痕试验检测miR-133b转染后COV434细胞的迁移情况,MTT法检测miR-133b转染后COV434细胞的增殖情况。结果miR-133b作用于FOXL2 mRNA的3′UTR区域,阴性对照组与空白对照组比较,COV434细胞FOXL2基因mRNA和蛋白的表达水平均无统计学差异(P>0.05),而miR-133b转染组与阴性对照组和空白对照组比较,差异均有统计学意义(P<0.05),提示miR-133b能显著抑制FOXL2 mRNA和蛋白的表达。同时,miR-133b转染COV434细胞后亦能明显抑制COV434细胞的迁移与增殖。结论miR-133b可抑制COV434细胞FOXL2 mRNA和蛋白的表达,进而抑制COV434细胞的迁移和增殖。

Objective To establish an overexpression model of miR-133b in human ovarian granular tumor cells(COV434),and to explore its effect on FOXL2 gene expression,and study whether it can play a role in the migration and proliferation of COV434 cells.Methods Bioinformatics method was used to analyze whether miR-133b targeted the UTR region of FOXL2 gene.Based on the predicted result,miR-133b mimics targeting FOXL23'UTR were designed and synthesized.The miR-133b mimics were artificially synthesized and transfected in COV434 cells by using Lipofectamine 3000 Reagent,and the transfection time was determined by observing the fluorescence.After miR-133b mimics were transfected into human ovarian granular tumor cells,qRT-PCR was used to detect the expression level of FOXL2 gene mRNA in COV434 cells,and the expression level of FOXL2 protein was detected by Western Blot method.Scratch test was used to detect the cell migration ability.MTT assay was used to determine the effect of target miR-133b on COV434 cells proliferation.Results miR-133b acted on the 3'UTR region of FOXL2 mRNA.There were no significant differences in the expression levels of FOXL2 gene mRNA and protein in COV434 cells between the negative control group and the blank control group(P>0.05),while the differences between the miR-133b transfected group and the negative control group and the blank control group were statistically significant(P<0.05),suggesting that miR-133b could significantly inhibit the expression of FOXL2 mRNA and protein.At the same time,miR-133b significantly inhibited the migration and proliferation of COV434 cells after transfection of COV434 cells.Conclusion miR-133b could inhibit the expression of FOXL2 mRNA and protein in COV434 cells,and inhibit the migration and proliferation of COV434 cells by targeting FOXL2.