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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(bisulfite sequencing PCR,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365 bp,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。
HPV16-positive cervical cancer cells and RNAi were selected to study the relationship between CALCA methylation and HPV16-E7 oncogene protein expression. Construction of recombinant lentiviral siRNA expression vector, the establishment of stable expression of HPV16-E7-siRNA RNAi cell model. The genomic DNA of SiHa cells and RNAi cell model was used as the target, and the target gene fragment rich in CpG islands in the promoter region of CALCA gene was selected and analyzed by bisulfite sequencing PCR (BSP) E7 expression, CALCA gene methylation status of the degree of reversibility. The target fragment rich in CpG locus in the promoter region of CALCA gene was selected and its size was 365 bp with 19 CpG islands. Cytosine at 13 CpG sites was found to be methylated in the genomic DNA of SiHa cells 13/19) whereas all CpG sites have been methylated (0/19 site) in RNAi cell models expressing HPV16-E7-siRNA. This study demonstrated that CALCA gene promoter hypermethylation in cervical cancer cells is dependent on the expression of oncogenic protein HPV16-E7 at the cellular level, which laid an important material foundation for further study on the role of E7 protein and carcinogenesis.