摘要
目的:研究转录因子Sp1在肝癌细胞血管内皮生长因子(VEGF)转录调控中的作用。方法:构建VEGF启动子双报告基因质粒系统,脂质体转染至肝癌细胞株MHCC97H中,分析VEGF表达调控的主要作用区域及转录因子Sp1对VEGF转录活性的影响;采用凝胶电泳迁移率变动分析转录因子Sp1与VEGF启动子的结合活性;采用RNA干扰技术进一步分析转录因子Sp1对VEGF的转录调控作用。结果:应用VEGF全长启动子及5端缺失突变体报告基因分析发现VEGF启动子上88～-61区域是VEGF转录的最主要的调控区域;用点突变体报告基因技术发现此区域上的转录因子Sp1结合位点突变后VEGF表达明显下调;凝胶电泳迁移率变动分析证实Sp1可特异性结合于VEGF启动子上;Sp1特异性siRNA下调转录因子Sp1表达后发现VEGF的表达也随之明显下调。结论:转录因子Sp1与VEGF启动子特异性结合是VEGF转录调控最重要的机制,并进一步调控肝癌血管生成及转移复发,深入分析转录因子Sp1在肝癌中的表达将有利于探讨肝癌血管生成及转移复发机理。
Objective：To evaluate the role of transcription factors Sp1 in transcription control of Vescular endothelial growth factor（VEGF） in human hepatocellular carcinoma. Methods： Contructing reporter plasmids and mutagenesis containing VEGF promoter, transfecting to human hepatocellular carcinoma cell line MHCC97H, and analyzing the critical regions of VEGF transcriptional control, and the role of Sp1 on transcription control of VEGF. Electrophoretic mobility shift assays （EMSA） were used to determine the binding activity of Sp1, and knockdown the expression of Sp1 by RNAi to analyze the role of Sp1 expression in the transcription control of VEGF. Results： A series of 5＇ deletion mutants of VEGF promoter reporter gene based on the 2274-bp VEGF promoter were transfected into MHCC97H cells, and examined VEGF promoter activity. The result showed that the -109/-61 region contains essential regulatory elements. Point mutagenesis of transcription factor binding sites showed that eliminated the inhibitory effect of IFN-α on VEGF promoter activity, whereas such effect was not found in mutation of either AP-2 or Egr-1 sites. It was suggested that Sp1 binding sites in this region were responsible for VEGF promoter activity. Using RNA interference technique to reduce expression level, and found that VEGF promoter activity was almost eliminated. Conclusion：Transcription factor Sp1 binding VEGF promoter plays a crucial role in the transcription control of VEGF, and associate with metastatic potential of hepatocellular carcinoma.
出处
《中国临床医学》
北大核心
2007年第4期487-490,共4页
Chinese Journal of Clinical Medicine
基金
国家自然科学基金(30571800)
高等学校全国优秀博士学位论文作者专项资金(200263)
