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目的:以改进的银染RNAAP-PCR法分析原发性及转移性胃癌标本,鉴定和克隆胃癌转移相关基因。方法:以原发及转移灶胃癌标本总RNA为研究对象,首先以T12MN“锚定”引物进行逆转录合成cDNA第一链,再以10核苷酸任意引物进行PCR扩增,5%非变性聚丙烯酰胺凝胶电泳分离扩增产物后银染显示并回收差异条带,经RNA点杂交验证、克隆测序后进入GenBank数据库检验同源性。结果:经银染RNAAP-PCR法分析,获得系列差异表达片段。对其中MGA1(662bp)和PGA1(589bp)两个片段进行验证和测序,结果显示,MGA1与胃癌转移相关,并与编码人巨噬细胞集落刺激因子受体(macrophagecolony-stimulatingfactorreceptor,CSF-1R)的原癌基因c-fms100%同源;而PGA1则属胃癌转移抑制基因,与编码肿瘤转移抑制基因KAI-1完全同源。结论:银染RNAAP-PCR法适用于分析和克隆差异表达基因,具有快速、直观、假阳性率低等优点;提示CSF-1R及KAI-1与胃癌转移表型相关,为进一步研究CSF-1R功能提供了新的线索。
Objective: To analyze the primary and metastatic gastric cancer specimens with improved silver-stained RNAAP-PCR method and identify and clone metastasis-related genes in gastric cancer. METHODS: The primary RNAs of primary and metastatic gastric cancer specimens were used as the research object. Firstly, the first strand of cDNA was synthesized by T12MN “anchor” primers, and then the first strand was amplified by 10 nucleotides of any primers. 5% non-denatured After electrophoresis on polyacrylamide gels, silver staining was performed to reveal and recover the differential bands. After verification by RNA dot blot, the clones were sequenced and then entered into the GenBank database for homology analysis. Results: A series of differentially expressed fragments were obtained by silver staining RNAAP-PCR analysis. The two fragments of MGA1 (662bp) and PGA1 (589bp) were verified and sequenced. The results showed that MGA1 was related to the metastasis of gastric cancer and was associated with the encoding of human macrophage colony-stimulating factor receptor (CSF-1R). Proto-oncogene c-fms is 100% homologous; PGA1, however, is a metastasis suppressor gene of gastric cancer and is completely homologous to KAI-1, a tumor metastasis suppressor gene. Conclusion: The silver-stained RNAAP-PCR method is suitable for the analysis and cloning of differentially expressed genes, which has the advantages of rapid, intuitive, and low false-positive rates. It suggests that CSF-1R and KAI-1 are related to the metastatic phenotype of gastric cancer. To further study CSF-1R Features provide new clues.