结肠癌实质细胞长标签基因表达系列分析文库的建立及应用  

Construction and application of long serial analysis of gene expression library in parenchyma cells of colon cancer

作　　者：樊军卫[1] 唐华美[2] 严东旺[1] 王兆文[1] 王晓亮[1] 彭志海[1] 

机构地区：[1]上海交通大学附属第一人民医院普外科,上海200080 [2]上海交通大学附属第一人民医院病理科,上海200080

出　　处：《肿瘤》2010年第6期505-509,共5页Tumor

基　　金：国家高技术研究发展计划(863计划)项目(编号:2007AA022003);上海市科学技术委员会科研计划项目(编号:09411963500)

摘　　要：目的:应用长标签基因表达系列分析(long serialan alysis of gene expression,LongSAGE)技术定量分析结肠癌实质细胞转录组,并筛选肿瘤相关基因。方法:应用激光捕获显微切割(laser capture microdissection,LCM)、RNA提取和线性扩增方法得到结肠癌实质细胞及其相应正常结肠上皮细胞的反义RNA(antisense RNA,aRNA)样品,构建LongSAGE文库;然后应用SAGE2000软件对所得序列文本进行分析,筛选肿瘤相关基因;最后采用RT-PCR方法验证该差异表达基因。结果:结肠癌细胞LongSAGE文库标签体总数为50542个,识别出基因总数为16497个。正常结肠细胞LongSAGE文库标签体总数为50124个,识别出基因总数为16417个。LongSAGE文库筛选出结肠癌差异表达基因705个(P<0.05)。转化生长因子β诱导基因(transforming growth factor β-induced gene,TGFBI)为LongSAGE文库筛选出的结肠癌相关基因,RT-PCR检测验证了其在结肠癌组织标本中表达上调。结论:LCM-LongSAGE是对结肠癌实质细胞进行定量转录组研究的可行流程。Objective：To quantitatively analyze the transcriptome profiling of microdissected colon carcinoma tissues by long serial analysis of gene expression （LongSAGE） technique and screen the tumor-associated genes.Methods：Parenchyma cells in colon carcinoma tissues and corresponding normal colonic epithelium were microdissected by using laser capture microdissection （LCM） technique. RNA was extracted from the cells. Antisense RNA （aRNA） was obtained after linear amplification and was used for construction of the LongSAGE library. The sequence data in the library were analyzed using SAGE2000 software. Tumor-associated genes were screened from the LongSAGE library and were detected by reverse transcriptase PCR（RT-PCR）.Results：50 542 tags were generated in the LongSAGE library of colon cancer cells and 16 497 genes were identified; 50 124 tags were generated in the LongSAGE library of normal colonic epithelium and 16 417 genes were identified . There were 705 genes with differential expression were screened between normal tissues and tumor tissues （P〈0.05） . Transforming growth factor beta-induced gene （TGFBI） was one of the differentially expressed genes screened by LongSAGE technique . The up-regulated expression of TGFBI gene was confirmed by RT-PCR in colon cancer.Conclusion：LCM-LongSAGE is a feasible method for quantitative analysis of the transcriptome profiling of parenchyma cells of colon carcinoma.

关 键 词：结肠肿瘤 基因表达谱 序列分析 RNA 显微切割 基因表达系列分析 基因 TGFBI 

分 类 号：R735.35[医药卫生—肿瘤]