miR-497-5p在肝细胞癌组织中的表达水平及其对肝癌细胞增殖的影响

Expression of miR-497-5p in hepatocellular cancerous tissues and its influence on proliferation of hepatic carcinoma cells

目的探讨miR-497-5p在肝细胞癌组织中的表达水平及其对肝癌细胞增殖的影响。方法(1)选取46例肝细胞癌患者的肝细胞癌组织和癌旁组织,以及正常肝细胞株(THLE-3细胞)、人肝癌细胞株(SMCC7721细胞、MHCC97H细胞、LM3细胞、HepG2细胞、Hep3B细胞),检测miR-497-5p表达水平;取上述组织,检测胰岛素样生长因子1受体(IGF-1R)mRNA表达水平。比较不同miR-497-5p表达水平的肝细胞癌患者的临床病理特征。(2)将LM3细胞和HepG2细胞分为阴性对照组、miR-497-5p模拟物组和miR-497-5p+IGF-1R组进行实验。将miR-497-5p对照物、miR-497-5p模拟物分别转染至阴性对照组、miR-497-5p模拟物组细胞,将过表达IGF-1R慢病毒、miR-497-5p模拟物先后转染至miR-497-5p+IGF-1R组细胞。转染后48 h,检测3组细胞增殖情况,以及阴性对照组、miR-497-5p模拟物组细胞的IGF-1R mRNA表达水平。(3)应用TargetScan在线预测软件和双荧光素酶报告实验分析miR-497-5p与IGF-1R的关系。分析肝细胞癌组织中miR-497-5p表达水平与IFG-1R mRNA表达水平的相关性。结果(1)肝细胞癌组织中miR-497-5p表达水平低于癌旁组织,且各人肝癌细胞株的miR-497-5p表达水平均低于正常肝细胞株(均P<0.05)。相比于miR-497-5p高表达组,低表达组肿瘤大小>5 cm的患者比例、静脉侵犯的患者比例、TNM分期为Ⅱ~Ⅲ期的患者比例更高(均P<0.05)。(2)miR-497-5p模拟物组LM3细胞培养48 h、72 h、96 h后的吸光度值均小于阴性对照组,HepG2细胞培养24 h、48 h、72 h、96 h后的吸光度值均小于阴性对照组(均P<0.05)。无论是LM3细胞还是HepG2细胞,miR-497-5p模拟物组的IGF-1R mRNA表达水平均低于阴性对照组(均P<0.05)。miR-497-5p+IGF-1R组LM3细胞培养48 h、72 h、96 h后的吸光度值均大于miR-497-5p模拟物组,miR-497-5p+IGF-1R组HepG2细胞培养72 h、96 h后的吸光度值均大于miR-497-5p模拟物组(均P<0.05)。(3)生物信息学预测和双荧光素酶报告实验结果提示IGF-1R是miR-497-5p的直接作用靶基因。肝细胞癌组织中IGF-1R mRNA表达水平高于癌旁组织,且与miR-497-5p表达水平呈负相关(均P<0.05)。结论miR-497-5p在肝细胞癌组织和人肝癌细胞系中表达下调,过表达miR-497-5p可以抑制肝癌细胞的增殖能力。miR-497-5p可能通过靶向调节IGF-1R抑制肝癌细胞的增殖,miR-497-5p可能是一种潜在的治疗肝癌的新靶点。

Objective To explore the expression of miR-497-5p in hepatocellular cancerous tissues and its influence on proliferation of hepatic carcinoma cells.Methods(1)The hepatocellular cancerous and paracancerous tissues of 46 patients with hepatocellular carcinoma,as well as normal hepatocyte strain(THLE-3 cell),human hepatic carcinoma cell strains(SMCC7721 cell,MHCC97H cell,LM3 cell,HepG2 cell,and Hep3B cell)were selected to detect the expression of miR-497-5p;furthermore,the aforementioned tissues were obtained to detect mRNA expression of insulin-like growth factor 1 receptor(IGF-1R).The clinicopathological features were compared between hepatocellular carcinoma patients with various miR-497-5p expressions.(2)LM3 and HepG2 cells were assigned to negative control group,miR-497-5p mimics group,or miR-497-5p+IGF-1R group for experiment.The miR-497-5p controls and mimics were transfected into cells of the negative control group and miR-497-5p mimics group,respectively,and over-expressed IGF-1R lentivirus and miR-497-5p mimics were transfected successively into cells of the miR-497-5p+IGF-1R group.After 48 hours of transfection,the cell proliferation of the 3 groups,and IGF-1R mRNA expression of cells in the negative control group and the miR-497-5p mimics group were detected.(3)The TargetScan online predictor and dual luciferase reporter assay were employed to analyze the relation between miR-497-5p and IGF-1R.The correlation of miR-497-5p expression with IFG-1R mRNA expression in hepatocellular cancerous tissues was analyzed.Results(1)The miR-497-5p expression in hepatocellular cancerous tissues was lower than that in paracancerous tissues,and the miR-497-5p expression of various human hepatic carcinoma cell strains was lower than that of normal hepatocyte strain(all P<0.05).Compared with the high-expressed miR-497-5p group,the low-expressed group exhibited higher proportions of patients with tumor size>5 cm,with venous invasion,with TNM stage fromⅡtoⅢ(all P<0.05).(2)After 48,72,and 96 hours of LM3 cell culture,the miR-497-5p mimics group yielded a smaller absorbance value as compared with the negative control group,and after 24,48,72,and 96 hours of HepG2 cell culture,the miR-497-5p mimics group yielded a smaller absorbance value as compared with the negative control group(all P<0.05).In both LM3 and HepG2 cells,the miR-497-5p mimics group interpreted a lower IGF-1R mRNA expression as compared with the negative control group(all P<0.05).After 48,72,and 96 hours of LM3 cell culture,as well as after 72 and 96 hours of HepG2 cell culture,the miR-497-5p+IGF-1R group presented a larger absorbance value as compared with the miR-497-5p mimics group(all P<0.05).(3)The results of bioinformatics predictor and dual luciferase reporter assay suggested that IGF-1R was the direct effect target gene of miR-497-5p.The mRNA expression of IGF-1R in hepatocellular cancerous tissues was higher than that in paracancerous tissues,and negatively correlated with the expression of miR-497-5p(all P<0.05).Conclusion The expression of miR-497-5p is down-regulated in hepatocellular cancerous tissues and in human hepatic carcinoma cell lines,and over-expression of miR-497-5p can inhibit the proliferation of hepatic carcinoma cells.MiR-497-5p may inhibit proliferation of hepatic carcinoma cells by targetell regulation of IGF-1R,and it may be a potential novel target for the treatment of hepatic carcinoma.