宫颈癌中CALCA基因甲基化与HPV16-E7致癌蛋白表达的依存关系

 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-E 7 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。

Association of CALCA Gene Methylation and HPV16-E7 Oncoprotein Expression in Cervical Cancer

The dependence of the CALCA gene promoter hypermethylation on the HPV16-E7 oncoprotein expression is investigated by the RNAi technique and using the HPV16-positive SiHa cervical carcinoma cells as the target cells. A recombinant lentiviral siRNA expression vector is constructed, and an RNAi cell model stably expressing the HPV16-E7-siRNA is established. After the extraction of the genomic DNA from the SiHa cells and the RNAi cell model, the reversibility of the CALCA gene promoter hypermethylation induced by the RNAi inhibition of the HPV16-E7 oncogene expression is analyzed by the PCR amplification, the subsequent cloning and the sequencing of a CpG-rich target fragment in the CALCA gene promoter. A 365 bp CpG-rich sequence selected in the CALCA promoter region is found as the target fragment containing 19 CpG islands, among which the cytosine of 13 CpG sites is methylated in the genomic DNA of the SiHa cells (13/19 CpG sites), whereas all methylations are fully reversed in the RNAi cell model expressing the HPV16-E7-siRNA (0/19 CpG sites). It is shown that the CALCA gene promoter hypermethylation is directly dependent on the HPV16-E7 oncoprotein expression in the cervical carcinoma cells, and the foundation for the role study and the carcinogenic mechanism of the E7 protein is established.