MEK1表达载体的构建及其对肝癌细胞ERK通路活性的影响  

Construction and identification of recombinant expression vector contained MEK1 and its effect on ERK activity in hepatocellular carcinoma cells

作　　者：梁容瑞[1,2] 周蕊[2,3] 李宗芳[2,3,4] 李宝华 陈昆仑[2,3] 王志东[2,3] 姬媛媛 杨军 黄辰[3,4] 

机构地区：[1]苏州大学附属第一医院肿瘤科,江苏苏州215006 [2]西安交通大学医学院第二附属医院普通外科干部病房,陕西西安710004 [3]生物诊断治疗国家地方联合工程研究中心,陕西西安710004 [4]西安交通大学医学院环境与疾病相关基因教育部重点实验室,陕西西安710077

出　　处：《西部医学》2013年第10期1447-1450,共4页Medical Journal of West China

基　　金：教育部"长江学者和创新团队发展计划"资助(PCSIRT:1171)

摘　　要：目的构建pcDNA3.1(+)-MEK1真核表达载体,体外转染人肝癌MHCC97H细胞,观察MEK1在肝癌细胞中的表达及其对ERK通路活性的影响。方法应用RT-PCR扩增出MEK1基因插入pcDNA3.1(+)中,形成重组载体pcDNA3.1(+)-MEK1。经测序确认后,利用LipofectamineTM 2000将重组载体转染MHCC97H肝癌细胞,使用含G418的培养基进行筛选;将肝癌细胞分为不转染组、转染空载体组和转染重组载体组,运用Western-blot方法检测MEK1、p-MEK1、ERK1/2、p-ERK1/2在各组细胞中的表达并绘制各组细胞的生长曲线对比其增殖能力。结果重组载体pcDNA3.1(+)-MEK1酶切鉴定电泳条带大小正确,测序结果经Blast比对与人MEK1基因开放性读码框100%吻合;重组载体载体转染肝癌细胞后MEK1表达水平较不转染及转染空载体载体组细胞显著升高,且磷酸化的MEK1及ERK1/2水平也明显升高,转染重组载体载体的肝癌细胞增殖能力得到显著增强。结论成功构建了真核表达载体pcDNA3.1(+)-MEK1,过表达MEK1蛋白可提高肝癌细胞MAPK/ERK通路活性。Objective To construct the expression vector of pcDNA3.1（＋）-MEK1 and observe its effect on ERK activity human hepatocellular carcinoma cells MHCC97H. Methods The full length open reading frame of human MEK1 （MAP2K1） gene was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 （＋）. After identified by enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into human hepatocellular carcinoma cells using liposomes 2000, and the expression of MEK1, p-MEK1, ERK1/2 and p-ERK1/2 were detected by Western- blot. Growth curve assay was executed to evaluate the proliferation of cells post-transfection. Results Plasmid was di- gested, and the amplified fragments had 100% homology with the human MEK1 gene published in the Gene bank. Cells cultured in medium contained G418 with a concentration of 500mg/ml for 21 days could be indentified into transfected cells or not. The Western blot demonstrated a high expression of MEK1, p-MEK1 and p-ERK1/2 in MHCC97H cells af- ter trasfected by recombinant plasmid. A pro-proliferative effect due to recombinant plasmid was detected. Conclusion The eukaryotic expression vector pcDNA3.1（＋）-MEK1 was constructed successfully and the MEK1 protein can be ex- pressed successfully in human hepatocellular carcinoma cells MHCC97H, therefor induce a highly activation of ERK sig- naling in HCC cells.

关 键 词：MEK1 肝癌 MHCC97H 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]