微小RNA-202可通过转录后下调低密度脂蛋白受体相关蛋白6的表达抑制人肝癌细胞增殖

摘要

目的 观察微小RNA(miRNA,miR)-202是否通过转录后下调癌基因低密度脂蛋白受体相关蛋白6(LRP6)的表达而抑制人肝癌细胞(HCC)的增殖.方法 培养HCC细胞株QGY-7703、HepG2;采用实时定量聚合酶链反应(Real-time PCR)检测细胞周期蛋白(Cyclin) D1、MYC和LEF1 mRNA的表达;Western blot检测LRP6、Cyclin D1、MYC、β-连环蛋白(β-catenin)和磷酸化Rb蛋白的表达;通过转染小干扰RNA (siRNA)抑制肝细胞癌(HCC)细胞LRP6的表达;构建pGL3-LRP6-3'端非编码区域(3'-UTR)-荧光素酶质粒转染HCC细胞作荧光素酶检测;采用噻唑蓝(MTT)法、克隆形成实验及贴壁不依赖性生长能力测定法检测HCC细胞的增殖能力.结果 在QGY-7703、HepG2两种细胞株均显示,过表达miR-202可以下调LRP6的表达,而抑制miR-202可以上调LRP6的表达(P＜0.05);miR-202的过表达可以降低pGL3-LRP6-3'-UTR-荧光素酶报告基因中荧光素酶的活性,而抑制miR-202则可以增加其荧光素酶的活性(P＜0.05);miR-202可以显著下调受Wnt/β-catenin调控的下游基因Cyclin D1、MYC和LEF1的表达,抑制miR-202则可以显著上调这些基因的表达(P＜0.05);在miR-202过表达的HCC细胞,Cyclin D1、MYC、LEF1和磷酸化Rb蛋白的表达也显著下调,而抑制miR-202的表达,则可以上调这些蛋白的表达(P＜0.05);miR-202的表达与LRP6蛋白的表达水平呈显著负相关(r =0.575,P＜0.05);通过MTT法和克隆形成实验结果表明,沉默miR-202抑制组或阴性对照转染组细胞的LRP6,可以降低HCC细胞的生长速度和增殖(P＜0.05);贴壁不依赖性生长能力测定也显示,LRP6抑制对miR-202抑制组HCC细胞的增殖具有显著抑制作用(P＜0.05).结论 在HCC细胞,LRP6mRNA是miR-202的直接作用靶点;在miR-202发挥抑制HCC细胞增殖的作用中,LRP6抑制是必不可少的;LRP6可以作为HCC的潜在治疗靶点.%Objective To study whehter microRNA (miRNA,miR)-202 suppresses cell proliferation in human hepatocellular carcinoma (HCC) by downregulating low density lipoprotein receptor associated protein 6 (LRP6) post-transcriptionally.Methods The HCC cell lines QGY-7703 and HepG2 were cultured according to the manufacturer' s instruction.The relative expression levels of Cyclin D1,MYC and LEF1 mRNA were assessed by the real-time PCR analysis.The expression of c-Myc,Cyclin D1,β-catenin and phosphorylated Rb protein was assessed by Western blotting analysis.The small interfering RNA (siRNA) was used to inhibit the expression of LRP6 in HCC cells.The pGL3-LRP6-3' untranslated region (3'-UTR)-luciferase reporter was constructed and transfected into HCC cells to make luciferase assay.The methyl thiazol tetrazolium (MTT) assay,colony formation assay and anchorage-independent growth ability assay were used to assess the proliferation ability of HCC cells.Results The Western blotting analysis showed that LRP6 expression was downregulated by ectopic miR-202 and upregulated by miR-202 inhibitor in both QGY-7703 and HepG2 cells (P ＜ 0.05).The luciferase reporter assay showed that ectopic expression of miR-202 decreased the luciferase activity of the pGL3-LRP6-3 '-UTR-luciferase reporter,and suppression of miR-202 increased luciferase production (P ＜0.05).The downstream genes regulated by Wnt/β-catenin (Cyclin D1,MYC and LEF1) were all significantly downregulated by miR-202,and upregulated by the miR-202 inhibitor (P ＜ 0.05).Furthermore,the expression levels of c-Myc,Cyclin D1,β-catenin and phosphorylated Rb proteins were downregulated in miR-202 overexpressing cells,and upregulated in cells transfected with the miR-202 inhibitor (P ＜ 0.05).There was an obviously negative correlation between the expression of miR-202 and the LRP6 protein levels (r =0.575,P ＜ 0.05).Silencing LRP6 in miR-202 inhibitor or NC transfected cells decreased the growth rate and proliferation of cells,as assessed by MTT and colony formation assays (P ＜ 0.05).The anchorage-independent growth ability of miR-202-inhibited cells was also significantly suppressed in response to LRP6 inhibition (P ＜ O.05).Conclusion Our results suggested that LRP6 mRNA is a direct target of miR-202 in HCC cells.And the depletion of LRP6 is essential for miR-202-mediated inhibition of proliferation of HCC cells.