肝癌组织EMP1表达生物学意义

Expression and effect of EMP1 on cell biology in liver carcinoma

目的探讨上皮膜蛋白(epithelial membrane protein-1,EMP1)在肝癌组织中的表达水平及过表达对肝癌细胞生物表型的影响。方法采用免疫组织化学方法、蛋白质印迹法检测唐山市人民医院2008-01-01-2012-12-30 65例肝癌组织及35例癌旁肝脏组织中的表达情况(距其癌组织边缘≥2cm,镜下未见癌浸润)。采用慢病毒转染建立EMP1过量表达的肝癌HepG2细胞株。荧光定量PCR及蛋白质印迹法检测EMP1转染后肝癌HepG2细胞株中EMP1表达含量的变化。MTT法、流式细胞术及Transwell检测EMP1过量表达对肝癌细胞增殖、细胞凋亡及细胞侵袭转移的影响。结果免疫组织化学结果表明,EMP1蛋白在肝癌组织中的表达阳性率为32.3%(21/65),明显低于癌旁肝脏组织的85.7%(30/35),χ2=26.118,P<0.001。蛋白质印迹法检测结果表明,EMP1蛋白在肝癌组织的表达相对量(0.257±0.022)较癌旁肝脏组织(0.863±0.086)明显降低,两者比较差异有统计学意义,t=11.824,P<0.001。EMP1蛋白表达水平与肝癌有无淋巴结转移、临床分期、组织分级以及血行转移有关(P<0.05),而与肝癌患者年龄、性别、病理类型及T分期无关,P值均>0.05。EMP1高表达的肝癌细胞其增殖能力明显减弱,细胞凋亡及侵袭转移能力明显降低,Caspase-9蛋白表达上调,而血管内皮生长因子-C(vascular endothelial growth factor C,VEGFC)与基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白的表达量明显下调,P<0.05。结论肝癌组织中EMP1蛋白表达减低,可能通过调控Caspase-9及VEGFC蛋白表达影响肝癌细胞增殖、凋亡与转移的生物学过程。

OBJECTIVE To analyze the expression of epithelial membrane protein-1 （EMP1） in liver carcinoma and the biological effect in its ceil line by EMP1 overexpression. METHODS Immunohistochemistry and western blot were used to analyze EMP1 protein expression in 65 cases of liver cancer and 35 cases of paraneoplastic tissues collected from our hospital before December 30,2012. The relationship between EMP1 expression and clinical factors was studied. EMP1 lentiviral vector and empty vector were respectively transfected into liver cancer HepG2 cell line. Quantitative real-time RT-PCR （qRT-PCR） and western blot were used to detect the mRNA level and protein of EMP1. MTT assay,cell apop- tosis and invasion assays were also conducted to observe the biological influence of the up-regulated expression of EMP1 which might be found on HepG2 cell. RESULTS Immunohistochemistry：The level of EMP1 protein expression was found to be significantly lower in liver cancer tissue （32.3M, 21/65） than that in paraneoplastic tissues （85.7%, 30/35, X2 = 26.118, P〈0.001）. Western blot：The relative amount of EMP1 protein in liver cancer tissue （0.257± 0. 022） was found to be significantly lower than that in paraneoplastic tissues （0. 863 4±0. 086, t= 11; 824, P〈0. 001）. The level of EMP1 protein expression was not correlated with gender,age,T stages and pathological types （P〉0.05）, but it was correlated with lymph node metastasis, clinic stage, histological grade and blood metastasis （P〈0.05）. The results of biological function shown that HepG2 cell transfected EMP1 had a lower survival fraction,higher call apoptosis, and significant de- crease in migration and invasion,and higher Caspase-9 and lower VEGFC protein expression compared with HepG2 cell untransfected EMP1 （P〈0.05）. CONCLUSION EMP1 expression decreased in liver cancer,suggesting that EMP1 may play important roles as negative regulator to liver cancer HepG2 cell by promoting degradation of Caspase-9,VEGFC.