转录因子 YY1对口腔鳞癌细胞ITGB6基因表达的影响

摘要

AIM:To explore the role of transcription factor YY1 in the regulation of integrin β6 (ITGB6) gene expression in oral squamous cell carcinoma .METHODS: The distribution of the potential YY1 binding sites in ITGB6 promoter were predicted by bioinformatic methods .Series of 5′deletion of ITGB6 promoter luciferase reporter constructs containing potential YY1 binding sites were made and transfected into 293T cell line to detect the promoter activity .The binding activity of the transcription factor YY1 to ITGB6 promoter in the native chromatin environment was determined by chromatin immunoprecipitation (ChIP) assay.The role of the potential YY1 binding sites in the regulation of ITGB6 pro-moter activity was analyzed by substitution mutant analysis .The effect of YY1 over-expression on the mRNA expression of ITGB6 in oral squamous cell carcinoma cell line was measured by RT-PCR.RESULTS:Bioinformatics analysis revealed that there were several potential binding sites for YY 1 in the region of -421~-150 nt in the ITGB6 promoter.ChIP assay showed that transcription factor YY1 bound to the ITGB6 promoter region of -421~-150 nt in the native chromatin environ-ment.Substitution mutant analysis of potential YY 1 binding sites in ITGB6 promoter did not affect the promoter activity of ITGB6.The over-expression of YY1 in oral squamous cell carcinoma cells did not affect the ITGB 6 mRNA expression. CONCLUSION:The transcription factor YY1 binds to the region of -421~-150 nt in the ITGB6 promoter.However, it is not involved in the basic transcriptional regulation of ITGB6 gene in oral squamous cell carcinoma cells .%目的：研究肿瘤相关转录因子YY1对口腔鳞癌细胞整合素β6（integrin β6， ITGB6）基因表达调控的影响。方法：用生物信息学方法预测分布在ITGB6启动子区域的转录因子YY1潜在的结合位点，构建萤光素酶报告基因质粒，利用双萤光素酶报告基因系统检测ITGB6启动子片段的转录活性；利用染色质免疫沉淀技术检测在天然染色质条件下转录因子YY1与ITGB6启动子的结合情况；采用定点突变方法检测YY1结合位点对ITGB6启动子活性的影响；利用RT-PCR方法检测过表达转录因子YY1对口腔鳞癌细胞ITGB6 mRNA表达水平的影响。结果：ITGB6启动子-421～-150 nt区域存在多个转录因子YY1潜在的结合位点。在口腔鳞癌细胞的天然染色质中，转录因子YY1结合于ITGB6启动子-421～-150 nt区域；定点突变YY1潜在结合位点对口腔鳞癌细胞中ITGB6启动子活性无显著影响。另外，过表达的YY1对口腔鳞癌细胞ITGB6 mRNA的表达水平也无影响。结论：转录因子YY1在口腔鳞癌细胞中结合于ITGB6基因启动子-421～-150 nt区域，但对ITGB6基因的基础转录水平无影响。