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科技导报  2014 

宫颈癌中CALCA基因甲基化与HPV16-E7致癌蛋白表达的依存关系

DOI: 10.3981/j.issn.1000-7857.2014.10.011, PP. 63-67

Keywords: 降钙素相关肽基因,宫颈癌,RNA,干扰,HPV16,E7,基因

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Abstract:

选择HPV16阳性宫颈癌细胞和RNAi技术,研究CALCA基因甲基化与HPV16-E7致癌蛋白表达的依存关系。构建慢病毒siRNA重组表达载体,建立稳定表达HPV16-E7-siRNA的RNAi细胞模型。以SiHa细胞和RNAi细胞模型的基因组DNA为对象,选择CALCA基因启动子区富含CpG岛屿的目标片段,使用亚硫酸氢盐测序法(bisulfitesequencingPCR,BSP)筛查分析,研究RNAi抑制HPV16-E7表达后,CALCA基因甲基化状态的可逆性程度。选出CALCA基因启动子区富含CpG位点的目标片段,其大小为365bp,含有19个CpG岛屿,发现其中13个CpG位点的胞嘧啶在SiHa细胞基因组DNA中发生了甲基化(13/19),而在表达HPV16-E7-siRNA的RNAi细胞模型中,所有CpG位点的甲基化已发生逆转(0/19位点)。本研究从细胞水平证明了宫颈癌细胞内的CALCA基因启动子高甲基化对HPV16-E7致癌蛋白表达有依赖性,为进一步研究E7蛋白的作用及致癌机制奠定了重要的物质基础。

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