热休克蛋白75在肝癌细胞株中的表达及其在上皮间质转化中的作用  

Expression of Mortalin in human hepatoma-derived cell lines and its role in epithelial-mesenchymal transition

作　　者：陈静 刘文斌 荚卫东 许戈良 马金良 李建生 

机构地区：[1]安徽医科大学附属省立医院肝脏外科肝胆胰外科安徽省重点实验室,合肥230001

出　　处：《中华肝胆外科杂志》2014年第1期51-55,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金资助项目（81272398）;安徽省自然科学基金资助项目（12010402112）;安徽高校省级自然科学研究重点项目（KJ2011A171）

摘　　要：目的检测热休克蛋白75（Mortalin）在肝癌细胞株中的表达,探讨其在肝癌细胞上皮间质转化（EMT）中的作用.方法选择5株人肝癌细胞和1株正常肝细胞（L02）,采用蛋白印迹和实时定量PCR检测Mortalin的表达水平.应用RNA干扰技术（shRNA）抑制肝癌细胞株MHCC97H内源Mortalin基因的表达.采用MTT试验和流式细胞仪检测细胞活性,通过蛋白印迹和实时定量PCR检测Mortalin、E-cadherin和Vimentin的表达水平.实验设空白组、对照组和shRNA组.结果Mortalin在Hep3B、MHCC97H、HepG2和HCCLM3中表达,而在MHCC97L和L02中不表达.干扰24h后,绿色荧光显示质粒成功转进MHCC97H.MTT试验显示转染对三组细胞的毒害作用分别为0％、2.5％和3.5％,流式细胞仪检测三组细胞早期凋亡率分别为0.8％,4.5％和9.2％.这说明转染没有引起严重的细胞损伤.干扰48h后,蛋白印迹和实时定量PCR检测结果显示,shRNA明显抑制了Mortalin的表达.收集干扰24h、48h、72h和96h后的细胞,应用蛋白印迹和实时定量PCR分析Mortalin、E-cadherin和Vimentin的关系,发现Mortalin表达抑制伴随着E-cadherin表达升高和Vimentin表达降低.结论Mortalin的表达可能与肝癌细胞的转移表型有关,可促进肝癌细胞上皮间质转化.Objective To detect the expression of Mortalin in human hepatoma-derived cell lines and explore its effect on epithelial-mesenchymal transition in hepatocellular carcinoma （HCC） cell lines.Methods Six HCC cell lines and 1 normal liver cell line （L02） were chosen.The expression of Mortalin was detected using Western blot and real-time quantitative PCR （qPCR）.The endogenous gene expression of Mortalin was inhibited by RNA interference （shRNA）.Cell viability was detected using MTT assay and flow cytometry.The expression of Mortalin,E-cadherin and Vimentin were detected by Western blot and qPCR.The experiment was divided into three groups; blank,control,and shRNA.Results Mortalin was detected in Hep3B,MHCC97H,HepG2,and HCCLM3,but not in MHCC97L and L02.After 24 h transfection,GFP fluorescence showed that plasmid Mortalin shRNA was successfully transfected into MHCC97H cells.MTT assay indicated that cytotoxicity was 0%,2.5%,and 3.5% in the blank,control,and shRNA group respectively.Similarly,flow cytometric showed that early apoptosis rates were 0.8%,4.5%,and 9.2% in the blank,control,and shRNA group respectively.These results indicated that transfection did not cause severe cell damage.After 48 h of interference,Western blot and qPCR analysis showed that shRNA significantly inhibited the expression of Mortalin.Moreover,cells were collected after 24 h,48 h,72 h and 96 h of interference and analyzed for the relationship between Mortalin,E-cadherin and Vimentin by Western blot and qPCR.It was found that decreased expression of Mortalin was accompanied by elevated E-cadherin expression and reduced Vimentin expression.Conclusion Overexpression of Mortalin correlated with the metastatic phenotype of HCC cells and could promote epithelial-mesenchymal transition.

关 键 词：肝细胞癌 上皮-间质转化 波形蛋白 

分 类 号：R735.7[医药卫生—肿瘤]