Qin A D, Liu X X, Li J, Liu J, Li Y S
Research Institute of Liver Disease, the Fourth People's Hospital of Huai'an, Huai'an 223002, China.
Clinical Laboratory, the Fourth People's Hospital of Huai'an, Huai'an 223002, China.
Zhonghua Zhong Liu Za Zhi. 2018 Jun 23;40(6):406-411. doi: 10.3760/cma.j.issn.0253-3766.2018.06.002.
To investigate the effects of overexpression of microRNA-7 (miR-7) on the proliferation and invasion of HepG2 cells and the underlying mechanism . The 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. The relative expression level of miR-7 in HCC was 0.49±0.02, significantly lower than in ANT (1.21±0.05, <0.01). The level of miR-7 was significantly correlated the tumor volume, metastasis and prognosis of HCC patients (<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 (<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 (<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 (<0.01). The relative expression of Raf1 protein in HCC was 3.15±0.10, significant higher than in ANT (0.53±0.03, <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, <0.01). Furthermore, an negative correlation was observed between the levels of miR-7 and Raf1 in HCC tissues (<0.05). The 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 .
探讨微小RNA-7(miR-7)过表达对肝癌HepG2细胞增殖和侵袭的影响及其潜在机制。采用实时定量聚合酶链反应(qRT-PCR)检测肝癌(HCC)组织及癌旁正常组织(ANT)中miR-7和Raf1的相对表达水平。分析miR-7表达与HCC患者特征的关系。将细胞分为空白对照组、阴性对照组(NC)和miR-7模拟物转染组,采用Lipofectamine™2000将miR-7模拟物和NC转染至HepG2细胞。通过qRT-PCR检测miR-7的相对表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四氮唑溴盐(MTT)法检测HepG2细胞的增殖能力。采用Transwell法检测HepG2细胞的侵袭能力。通过TargetScan预测miR-7的靶基因,并采用双荧光素酶报告基因检测法验证miR-7与Raf1 3'非翻译区(UTR)的结合作用。采用蛋白质免疫印迹法检测肝癌组织、正常组织及miR-7模拟物转染的HepG2细胞中Raf1蛋白的表达。采用Pearson相关分析确定miR-7和Raf1 mRNA水平的相关性。HCC中miR-7的相对表达水平为0.49±0.02,显著低于ANT(1.21±0.05,P<0.01)。miR-7水平与HCC患者的肿瘤体积、转移及预后显著相关(P<0.05)。miR-7模拟物转染的HepG2组中miR-7的相对表达水平为12.67±0.40,显著高于空白组(P<0.01)。与空白组相比,miR-7模拟物转染组在转染后48小时和72小时的A值及侵袭能力显著下调(P<0.01)。与miR-7 NC组相比,miR-7模拟物转染组中野生型Raf1报告基因的荧光素酶活性显著降低(P<0.01)。HCC中Raf1蛋白的相对表达为3.15±0.10,显著高于ANT(0.53±0.03,P<0.01)。miR-7模拟物转染组中Raf1蛋白的相对表达为0.24±0.01,显著低于miR-7 NC组(0.98±0.02,P<0.01)。此外,HCC组织中miR-7和Raf1水平呈负相关(P<0.05)。HCC中miR-7表达显著降低,且与HCC患者的不良生存呈负相关。miR-7过表达可通过下调Raf1抑制肝癌HepG2细胞的增殖和侵袭能力。