miR-196a在肝癌细胞中的表达及其促增殖作用

目的: 探讨miR-196a在肝癌组织的表达水平和对肝癌Hep3B细胞增殖的影响,阐明其促进肝癌细胞增殖的可能机制。方法: 收集20例肝癌组织及对应癌旁组织,培养人正常肝细胞株L02及肝癌细胞株SMMC-7721、HepG2和Hep3B,采用qRT-PCR法检测miR-196a mRNA在肝癌组织和对应癌旁组织及肝癌细胞株中的表达水平。取处于对数生长期人肝癌Hep3B细胞随机分为对照组、miR-196a mimics转染组和miR-196a inhibitors转染组,MTT法检测各组Hep3B细胞增殖活力,qRT-PCR法检测各组Hep3B细胞miR-196a的表达,流式细胞术检测各组Hep3B细胞周期变化,qRT-PCR和Western blotting法检测各组Hep3B细胞miR-196a潜在靶点叉头转录因子(FOXO1)的表达水平。结果: 肝癌组织中miR-196a mRNA表达水平明显高于相应癌旁组织(P<0.05);miR-196a mRNA在人肝癌SMMC-7721、HepG2和Hep3B细胞中的表达水平明显高于人正常肝L02细胞(P<0.05)。与对照组比较,miR-196a mimics转染组Hep3B细胞中miR-196a mRNA表达水平明显升高(P<0.05),而miR-196a inhibitors转染组Hep3B细胞中miR-196a mRNA的表达水平明显降低(P<0.05)。与对照组比较,miR-196a mimics转染组Hep3B细胞增殖率明显升高(P<0.05), miR-196a inhibitors转染组Hep3B细胞增殖率明显降低(P<0.05),且呈时间依赖性。与对照组比较,转染24 h时,miR-196a inhibitors转染组Hep3B细胞G₀/G₁期细胞百分率明显升高(P<0.05),S期细胞百分率明显降低(P<0.05)。与对照组比较, miR-196a mimics转染组FOXO1 mRNA和蛋白表达水平明显降低(P<0.05)。结论: miR-196a可能通过FOXO1促进肝癌细胞的增殖,提示miR-196a可能成为肝癌诊断和治疗的新靶点。

Expression of miR-196a in liver cancer cells and its promotion effect on proliferation

Objective To explore the expression level of miR-196a in liver cancer tissue and the influence of miR-196a in the proliferation of liver cancer Hep3B cells, and to clarify the possible mechanism of its promotion effect on the poliferation of liver cancer cells. Methods Twenty pairs of fresh surgical specimens of liver cancer and adjacent tissues were collected.The normal liver cells L02 and liver cancer cells SMMC-7721, HepG2, and Hep3B were cultured.The expression levels of miR-196a in liver cancer tissue, adjacent tissue and liver cancer cells were detected with qRT-PCR.The human liver cancer Hep3B cells at the logarithmic phase were randomly divided into control and miR-196a mimics and miR-196a inhibitors groups; the cell viability was analyzed by MTT assay and the miR-196a expression in the Hep3B cells after transfection was detected by qRT-PCR.The cell cycle distribution was determined by flow cytometry.The expression of FOXO1, one of the potential targets of miR-196a, was determined using qRT-PCR and Western blotting method. Results The expression level of miR-196a in liver cancer tissue was significantly higher than that in adjacent tissue (P<0.05).The expression levels of miR-196a in hunan liver ceancer cells SMMC-7721, HepG2, and Hep3B, were significantly higher than that in human normal liver L02 cells(P<0.05).Compared with control group, the expression level of miR-196a mRNA in the Hep3B cells in miR-196a mimics group was significantly increased(P<0.05), but the miR-196a mRNA expression level in the Hep3B cells in miR-196a inhibitors group was signficantly decreased(P<0.05).Compared with control group, the proliferation rate of the Hep3B cells in miR-196a mimics group was significantly increased(P<0.05), but the proliferation rate in miR-196a inhibitors group was significantly decresed(P<0.05) in a time-dependent manner.Compared with control group, the ratio of the Hep3B cells at G₀/G₁ phase 24 h after transfection in miR-196a inhibitors group was significantly increased, but the ratio of cells at S phase was obvionsly decreased(P<0.05).Compared with control group, the mRNA and protein expression levels of FOXO1 in miR-196a mimics group were significantly decreased(P<0.05). Conclusion miR-196a may promote the proliferation of live cancer cells through FOXO1, which indicates that miR-196a may become a new target for dignosis and treatment of liver cancer.