microRNA-200a抑制肝细胞癌的进展及调控索拉非尼治疗效果的研究

Study on the effect of microRNA-200a on the progression of hepatocellular carcinoma and the therapeutic effect of sorafenib

目的研究microRNA-200a(miR200a)影响肝细胞癌的进展及调控索拉非尼治疗效果。方法构建人肝癌细胞miR200a稳转细胞株:miR200a过表达、对照组(Control)、miR200a敲低。采用qRT-PCR法检测各转染细胞株miRNA表达量,MTT的方法检测细胞活力,Western blot分析各目标蛋白表达量;Transwell法检测肝癌细胞迁移、侵袭能力。转染细胞株添加索拉非尼,复测MTT及Western blot,验证miR200a调控索拉非尼对细胞的作用和相应蛋白表达量变化,SPF级BALB/c-nu雄鼠18只,随机分为3组,每组6只。每组皮下种植miR200a过表达、Control、miR200a敲低细胞,待成瘤后每组裸鼠每天注射索拉非尼,连续14 d,处死裸鼠,取出瘤体,计算肿瘤体积,免疫组化验证瘤体目标蛋白表达及定位。结果上调miR200a不仅可以抑制人肝癌细胞的细胞活力及迁移侵袭能力,还可以降低SIRT1的表达。并且上调miR200a明显减小裸鼠的肿瘤体积。结论上调miR200a可以负性调控人肝癌细胞的增殖和迁移能力,并增加索拉非尼的药物疗效,这种作用可能是通过调控SIRT1表达来完成。

Objective To study the effect of microRNA-200a(miR200a)on the progression of hepatocellular carcinoma and the therapeutic effect of sorafenib.Methods The stable transformation cell lines of human hepatocellular carcinoma miR200a were constructed:miR200a overexpression,Control and miR200a knockdown.The expression of miR200a in each transfected cell line was detected by qRT-PCR,the expression of protein was detected by Western blot method,the cell′s vitality was detected by MTT method,and the ability of cell migration and invasion was detected by transwell method.After adding sorafenib to the transfected cell line,the effect of miR200a on the regulation of sorafenib on cells and the changes of cell vitality and corresponding protein expression were detected by MTT and Western blot.Eighteen SPF male BALB/c-nu mice were randomly divided into 3 groups,with 6 mice in each group.miR200a overexpression,Control and miR200a knockdown cells were subcutaneously implanted in each group.After tumor formation,each group of nude mice were injected with sorafenib every day for 14 days.The nude mice were killed and the tumor volume was calculated.The expression and localization of tumor target protein were verified by immunohistochemistry.Results miR200a could inhibit the cell viability and migration ability of hepatocellular carcinoma cells,and reduce the expression of SIRT1 at the same time.MiR200a significantly reduced the tumor volume of nude mice.Conclusion miR200a can inhibit the proliferation and migration of human hepatocellular carcinoma cell line and increase the efficacy of sorafenib,which may be achieved by regulating the expression of SIRT1.