应用寡核苷酸芯片鉴别肺鳞癌Ⅰb期特异相关基因

Identification of stageⅠb specific related genes in lung squmous cell cancer by oligonucleotide array

目的建立Ⅰb和Ⅲa期肺鳞癌的基因表达谱并筛选肺鳞癌Ⅰb期特异相关基因。方法收集肺正常、临床Ⅰb和Ⅲa期肺鳞癌组织,分别组成样品池,提取其总RNA并制备cDNA,合成生物素标记的cRNA探针,与人U133A寡核苷酸基因芯片进行杂交,以MSV5.0软件对其结果进行分析处理,在Affymetrix数据库进行信息查询,并以逆转录聚合酶链反应(RTPCR)验证芯片检测结果。结果按照多组平行比较的原则进行共筛选,以信号值对数比(signallogratio,SLR)均大于1为筛选标准,肺鳞癌差异表达基因:Ⅰb期共计1764个,其中上调571个,下调1193个;Ⅲa期共计554个,上调128个,下调426个。相对于Ⅲa期基因表达谱,Ⅰb期特异的差异表达基因共有1329个,其中上调482个,下调847个,对其进行生物学功能分类,其中代谢相关480个,信号转导227个,细胞增殖136个,免疫相关136个,细胞黏附94个,转录调控88个,细胞周期86个,细胞骨架73个,细胞分化45个,细胞凋亡42个,胞外基质31个。FOXM1和TNXB基因的RTPCR检测结果与基因芯片检测结果一致。结论在全基因组范围内建立了Ⅰb和Ⅲa期肺鳞癌的基因表达谱并筛选了肺鳞癌Ⅰb期特异相关基因,为进一步的癌变机制研究和鉴定肺鳞癌早期诊断分子标记物奠定了基础。

Objective To establish gene expression profile of stage Ⅰ b and Ⅲ a primary lung squmous cell cancer （SCC） within whole genome and identify genes specifically expressed in stage Ⅰb of SCC. Methods Total RNA was extracted from the normal, stage Ⅰ b and Ⅲ a lung SCC tissue, cRNA probes prepared from total RNA were hybridized with pretreatment oligonucleotide chip containing 22215 genes. The resultant data were treated with MSV 5.0 software and looked up on the affymetrix website. RTPCR examination were used to validated the results from chip analysis. Results Comparing with the normal lung, difference genes expressed in Ⅰ b SCC .are totally 1764, amomg which 571 were upregulated and 1193 were downregulated, and in stage Ⅲ a, they are 554, 128 and 426 genes respectively. Genes specifically expressed in stage Ⅰ b were totally 1329, including 482 upregulation genes and 847 downregulation genes, which were classified into different category which included 480 metabolism related genes, 227 signal transduction genes, 136 cell proliferation genes, 136 immune related genes, 94 cell adhere genes, 88 transcription regulation genes, 86 cell cycle genes, 73 cytoskeleton genes, 45 differentiation genes, 42 apoptosis and 31 extracellular matrix genes. RT-PCR examination of FOXM1 and TNXB genes was consistent with the analysis of gene chip. Conclusion Stage Ⅰ b and Ⅲ a Gene expression profile of primary SCC within the whole genome were set up and genes specificially expressed in stage Ⅰ b of SCC were identified, which lay a foundation for further research on carcinogenesis mechanism and identifying new markers of early diagnosis.