MiR-125b-5p抑制卵巢癌细胞的迁移和侵袭:基于靶向下调CD147的表达

MiR-125b-5 suppresses ovarian cancer cell migration and invasion by targeted downregulation of CD147

目的探讨miR-125b-5p靶向调控细胞外基质金属蛋白诱导因子(CD147)表达对卵巢癌细胞生物学行为的影响及作用机制。方法应用荧光实时定量PCR技术(RT-qPCR)检测卵巢癌组织和癌细胞株中miR-125b-5p和CD147 mRNA的表达;构建过表达miR-125b-5p的SKOV3细胞或低表达miR-125b-5p的HO8910细胞。CCK-8实验、克隆形成实验、Transwell实验检测过表达或低表达miR-125b-5p对卵巢癌细胞的增殖、迁移和侵袭能力的影响;使用Starbase(http://starbase.sysu.edu.cn/)生信软件预测miR-125b-5p与CD147之间的潜在互补结合位点并使用双荧光素酶报告基因实验进行验证其靶向关系;体外培养SKOV3细胞,分别转染mimic NC、miR-125b-5p mimic、miR-125b-5p mimic和pcDNA3.1、miR-125b-5p mimic和pcDNA3.1-CD147,转染后分为mimic NC组、miR-125b-5p mimic组、miR-125b-5p mimic组+pcDNA3.1组、miR-125b-5p mimic+pcDNA3.1-CD147组,应用RT-qPCR和Westemblot实验检测各组中miR-125b-5p水平,CD147蛋白和mRNA水平;应用CCK-8实验、Transwell实验检测各组卵巢癌细胞的增殖、迁移和侵袭能力。结果RT-qPCR技术证实miR-125b-5p在癌组织和卵巢癌细胞株中均低表达,而CD147在癌组织和卵巢癌细胞株中均高表达(P<0.05);选择SKOV3和HO8910细胞进行转染,过表达miR-125b-5p后的卵巢癌细胞增殖、迁移、侵袭的能力均被抑制(P<0.05);Starbase生信软件预测及双荧光素酶报告基因实验证实miR-125b-5p和CD147存在一个靶向调控关系;在SKOV3细胞中转染过表达质粒和对照质粒,转染成功后,过表达miR-125b-5p组中miR-125b-5p的表达升高,而CD147的mRNA和蛋白表达明显下降(P<0.05)。过表达miR-125b-5p后再转染pcDNA3.1-CD147,CD147的mRNA和蛋白表达明显升高(P<0.05)。Transwell实验显示过表达miR-125b-5p后卵巢癌细胞的增殖、侵袭和迁移受到明显抑制,而过表达miR-125b-5p后再过表达CD147,卵巢癌细胞的增殖、侵袭和迁移的能力得到增强。结论miR-125b-5p通过负向调控CD147的表达,从而抑制卵巢癌细胞的迁移与侵袭。

Objective To investigate whether miR-125b-5p regulates biological behaviors of ovarian cancer cells by targeted regulation of CD147 expression.Methods RT-qPCR was used to detect the expression of miR-125b-5p and CD147 mRNA in ovarian cancer tissues and cancer cell lines.SKOV3 cells transfected with miR-125b-5p mimic and HO8910 cells transfected with miR-125b-5p inhibitor were examined for changes in proliferation,migration and invasion using CCK-8 assay,colony forming assay and Transwell assay.Starbase was used to predict the potential binding sites between miR-125b-5p and CD147,and double luciferase reporter gene assay was used to verify the targeting relationship.In SKOV3 cells,the effects of co transfection with miR-125b-5p mimic and pcDNA3.1-CD147(or pcDNA3.1)plasmid on cell proliferation,migration and invasion were assessed with CCK-8 assay and Transwell assay.Results The expression of miR-125b 5p was significantly lowered and that of CD147 was increased in both ovarian cancer tissues and ovarian cancer cell lines(P<0.05).Overexpression of miR 125b-5p in SKOV3 cells resulted in significantly suppressed cell proliferation,migration and invasion,while down regulation of miR-125b-5p in HO8910 cells promoted cell proliferation,migration and invasion.Bioinformatic analysis predicted that miR-125b-5p binds to CD147,which was confirmed by luciferase reporter gene assay.RT-qPCR and Western blotting showed that miR-125b-5p negatively regulated CD147 expression(P<0.05).In SKOV3 cells,the inhibitory effects of miR-125b-5p mimic on cell proliferation,invasion and migration were significantly attenuated by co-transfection of the cells with pcDNA3.1-CD147 plasmid.Conclusion miR-125b-5p inhibits the migration and invasion of ovarian cancer cells by negatively regulating the expression of CD147.