外源性S100A6蛋白对人结直肠癌细胞S100A6基因转录的影响及机制探讨  

Effect of Exogenous S100A6 Protein on The Level of S100A6 mRNA of Colorectal Carcinoma Cell Lines and Its Possible Mechanism

作　　者：孙晖[1] 李雪茹[1] 查何[1] 段亮[1] 袁世梅[1] 李欢[1] 李爱芳[1] 谷月[1] 周兰[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物工程杂志》2016年第1期14-22,共9页China Biotechnology

摘　　要：目的:探讨钙周期蛋白S100A6对人结直肠癌细胞系HCT116及SW480中S100A6基因转录的影响及机制。方法:首先采用原核表达方法制备重组人S100A6蛋白(recombinant human S100A6,rh S100A6)并鉴定;rh S100A6蛋白(终浓度为10μg/ml)、重组腺病毒Adsiβ-catenin(可表达β-catenin的siRNA)处理结直肠癌细胞HCT116、SW480及正常肠上皮细胞FHC,采用半定量反转录聚合酶链反应(reverse transcription and polymerase chain reaction,RT-PCR)及定量实时聚合酶链反应(quantitative real time polymerase chain reaction,q PCR)检测S100A6 mRNA的水平,采用RT-PCR检测细胞中E-cadherin mRNA水平,采用Western blot检测并比较HCT116及SW480细胞核中β-catenin蛋白的水平变化,采用细胞免疫荧光法检测细胞中β-catenin分布的变化。结果:成功制备rh S100A6蛋白;外源性rh S100A6蛋白上调HCT116及SW480细胞中S100A6 mRNA水平和细胞核中的β-catenin蛋白水平并促进β-catenin发生核移位,同时下调细胞中E-cadherin mRNA水平;Adsiβ-catenin与rh S100A6联合处理组的S100A6 mRNA水平明显低于单独rh S100A6处理组。所有差异均具有统计学意义。结论:胞外的S100A6蛋白增强结直肠癌细胞中S100A6基因的转录,其机制可能涉及β-catenin通路的激活。Objective：To investigate the effect of rh S100A6 on S100A6 mRNA of colorectal carcinoma cell lines HCT116 and SW480,and to explore its possible mechanism.Methods：Recombinant human S100A6（ rh S100A6） protien was prepared by prokaryotic expression.FHC,HCT116 and SW480 cells were treated with10μg / ml rh S100A6 and recombinant adenoviruses carrying human β-catenin siRNA（ Adsiβ-catenin）,semiquantitative reverse transcription-polymerase chain reaction（ RT-PCR） and quantitative real time polymerase chain reaction（ q PCR） was used to detect the expression of S100A6 mRNA,RT-PCR was used to detect the expression of E-cadherin mRNA,Western blot was used to detect the expression of β-catenin of nuclear protein in HCT116 and SW480 cells and immunofluorescence staining was used to detect the distribution of β-catenin.Results：rh S100A6 protein was prepared successfully and significantly increased the expression of S100A6 mRNA and nuclear β-catenin protein of HCT116 and SW480 cells,rh S100A6 treatment promoted β-catenin translocating from cytoplasm to nucleus in HCT116 and SW480 cells,at the same time,the E-cadherin mRNA level was decreased with the treatment of rh S100A6 protein.Compared with rh S100A6 treatment group,the expression level of S100A6 mRNA was decreased in co-treatment group by Adsiβ-catenin and rh S100A6.Conclusion：Extracellular rh S100A6 upregulate the experssion of S100A6 mRNA in colorectal carcinoma cell lines and its mechanism is partly involved in Wnt / β-catenin pathway.

关 键 词：结直肠癌 S100A6 Β-CATENIN 

分 类 号：Q819[生物学—生物工程]