肝细胞黏附分子对膀胱移行癌细胞基因表达谱的影响

摘要

目的 利用Affymetrix基因表达谱芯片探讨肝细胞黏附分子(hepaCAM)对膀胱移行癌细胞基因表达谱的影响及作用的分子机制.方法 运用Affymetrix Human plus 2.0全基因组芯片检测膀胱移行癌细胞株EJ细胞腺病毒-绿色荧光蛋白-hepaCAM感染组与腺病毒-绿色荧光蛋白感染组的基因表达谱,用SAM法进行差异基因分析、DAVID软件进行基因本体论分析及wikiPathway分析,并用RT-PCR和Western blot验证芯片结果.结果 hepaCAM能引起EJ细胞系基因表达谱的广泛变化,表达差异超过2倍以上的基因共2469个,其中上调基因1602个、下调基因867个.这些差异表达的基因功能主要涉及细胞周期、细胞增殖调节等方面.用RT-PCR对差异基因DNA修复蛋白,肝脏激酶B1和细胞周期蛋白D1进行验证,结果与基因芯片相符;进一步在3细胞株中用Western blot佐证DNA修复蛋白、肝脏激酶B1变化与芯片结果一致.结论 hepaCAM能够诱导EJ细胞基因表达谱的广泛变化,在基因水平通过多条通路作用于膀胱癌细胞.%Objective To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule ( hepaCAM) overexpression. Methods Affymetrix Human Genome U133 Plus 2. 0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein (GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells. Significant Analysis of Microarray (SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data. Results Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated. Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data. The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression. Conclusions HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.