微小RNA-7对肝癌HepG2细胞增殖和侵袭能力的影响

The effects of microRNA-7 on proliferation and invasion of hepatocellular carcinoma HepG2 cells

目的探讨过表达微小RNA－7（miR－7）对肝癌HepG2细胞体外增殖和侵袭能力的影响及调控机制。方法采用实时荧光定量PCR法检测肝癌组织和癌旁正常组织中miR－7 mRNA和Raf1 mRNA的表达，分析miR－7 mRNA的表达与肝癌患者临床病理特征的关系。将肝癌HepG2细胞分为空白对照组、miR－7阴性对照组和miR－7模拟物转染组，采用脂质体转染技术将miR－7模拟物和阴性对照瞬时转染HepG2细胞，以实时荧光定量PCR法检测miR－7 mRNA的表达，采用四甲基偶氮唑蓝（MTT）法检测不同时间点各组细胞的增殖活性，采用Transwell侵袭实验检测各组侵袭细胞数目。通过TargetScan在线预测miR－7作用的靶基因，采用荧光素酶报告基因实验验证miR－7对预测靶基因Raf1的3′非翻译区的靶向作用。采用Western blot法检测肝癌组织、相邻正常组织和各组细胞中Raf1蛋白的表达。采用Pearson法分析miR－7 mRNA与Raf1 mRNA表达的相关性。结果肝癌组织和相邻正常组织中miR－7 mRNA的相对表达水平分别为0.49±0.02和1.21±0.05，差异有统计学意义（P〈0.01）；miR－7 mRNA的表达水平与肿瘤大小、是否转移和预后有关（均P〈0.05）。miR－7模拟物转染组HepG2细胞中miR－7 mRNA的相对表达量为12.67±0.40，明显高于空白对照组（0.49±0.01，P〈0.01）。与空白对照组比较，转染48和72 h时，miR－7模拟物转染组的吸光度明显降低（P〈0.01），侵袭细胞数目明显减少（P〈0.01），野生型Raf1报告基因的荧光素酶活性明显降低（P〈0.01）。肝癌组织和相邻正常组织中Raf1蛋白的相对表达量分别为3.15±0.10和0.53±0.03，差异有统计学意义（P〈0.01）。miR－7阴性对照组和miR－7模拟物转染组HepG2细胞中Raf1蛋白的相对表达量分别为0.98±0.02和0.24±0.01，差异有统计学意义（P〈0.01）；肝癌组织中miR－7与Raf1的表达呈负相关（P〈0.05）。结论miR－7在肝癌组织中的表达明显降低，与肝癌患者的较差预后相关；miR－7能通过调控Raf1基因的表达抑制肝癌HepG2细胞的体外增殖和侵袭能力。

ObjectiveTo investigate the effects of overexpression of microRNA-7 （miR-7） on the proliferation and invasion of HepG2 cells and the underlying mechanism in vitro.MethodsThe relative expression levels of miR-7 and Raf1 in hepatocellular carcinoma （HCC） tissues and adjacent normal tissues （ANT） were detected by quantitative real time-PCR （qRT-PCR）. The relationship between the expression of miR-7 and the characteristics of HCC patients was analyzed. Cells were divided into blank control group, negative control （NC） group and miR-7 mimics transfected group, miR-7 mimics and NC were transfected into HepG2 cells by Lipofectamine？2000. The relative expression of miR-7 was detected by qRT-PCR. The proliferation ability of HepG2 cells was detected by 3-（4, 5-dimethyl-2-thiazolyl）-2, 5-diphenyl-2-H-tetrazolium bromide （MTT） assay. The invasion of HepG2 cells was detected by Transwell assay. The target genes of miR-7 were predicted by TargetScan and the binding effect of miR-7 on the 3′UTR of Raf1 was verified by dual luciferase reporter assay.The expressions of Raf1 protein in hepatocellular carcinoma tissues, normal tissues and miR-7 mimics transfected HepG2 cells was detected by Western blot. The correlation of the levels of miR-7 and Raf1 mRNA was determined by Pearson correlation analysis.ResultsThe relative expression level of miR-7 in HCC was 0.49±0.02, significantly lower than in ANT （1.21±0.05, P〈0.01）. The level of miR-7 was significantly correlated the tumor volume, metastasis and prognosis of HCC patients （P〈0.05）. The relative expression level of miR-7 in miR-7 mimics transfected HepG2 group was 12.67±0.40, significantly higher than that in blank group （P〈0.01）. Compared with the blank group, the A value and invasion ability of miR-7 mimics transfected group were significantly down-regulated at 48 hours and 72 hours after transfection （P〈0.01）. Compared with miR-7 NC group, the luciferase activity of wild-type Raf1 reporter gene in miR-7 mimics transfected group was significantly reduced （P〈0.01）. The relative expression of Raf1 protein in HCC was 3.15±0.10, significant higher than in ANT （0.53±0.03, P〈0.01）. The relative expression of Raf1 protein in miR-7 mimics transfected group was 0.24±0.01, significantly lower than in miR-7 NC group （0.98±0.02, P〈0.01）. Furthermore, an negative correlation was observed between the levels of miR-7 and Raf1 in HCC tissues （P〈0.05）.ConclusionsThe expression of miR-7 in HCC is significantly decreased and inversely correlated with poor survival of HCC patients. Overexpression of miR-7 can inhibit the proliferation and invasion ability of hepatocellular carcinoma cells HepG2 by downregulating Raf1 in vitro.