应用DNA芯片鉴定HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制

高危型人乳头瘤病毒（human papillomavirus,HPV）的E6基因在宫颈癌的发生中起关键作用，特异siRNA能有效抑制宫颈癌HeLa 细胞内HPV18 E6基因的表达，诱导肿瘤细胞凋亡.为进一步探讨HPV18 E6-siRNA诱导HeLa 细胞凋亡的分子机制，针对HPV18-E6基因设计siRNA序列，利用人源U6启动子为模板，经PCR表达框架法体外扩增，转染宫颈癌HeLa细胞抑制HPV18 -E6基因表达，从而诱导肿瘤细胞凋亡.对转染前后HeLa细胞总RNA样品进行荧光标记后，与Agilent Human 1A寡核苷酸芯片杂交、扫描、数据分析及标准化处理，确定表达差异的基因并经荧光定量PCR对部分基因进行验证，结合PANTHER数据分析系统，将这些基因按照生物学功能进行归类，查阅GenBank数据库及相关文献，对其结果进行深入分析及讨论.在检测的18 716个基因和EST中，共筛出差异表达基因359个，其中307个基因表达上调，52个基因表达下调，主要包括细胞周期相关基因CCNG1、p21；凋亡相关基因CASP4、CASP6、IGFBP3、DFFA；泛素蛋白酶解途径相关基因E6-AP、UBE2C；角化细胞分化相关基因KRT4、KRT6E、KRT18；抑癌基因RECK、VHL等.研究结果表明，HPV18 -E6基因抑制引起的细胞凋亡效应主要是通过P53信号途径和泛素蛋白酶解信号途径调节细胞周期相关基因和凋亡相关基因的表达，从而抑制HeLa细胞增殖、促进细胞凋亡.同时，抑癌基因的激活，角化细胞分化和免疫相关基因的表达上调，都说明了E6抑制后肿瘤细胞恶性转化程度的下降.

Study of apoptotic mechanism of HeLa cells induced by

The oncoprotein E6 of high-risk human papillomavirus (HPV) types promotes cell proliferation and contributes to carcinogenesis of HPV-positive cervical cancer cells. In the study, we used siRNA technology to silence E6 gene in HPV18 transformed human cervical cell line HeLa and determined the effects of E6 gene knockdown on the cell by using microarray-based gene expression profiling coupled with gene functional classification with bioinformatics methods. siRNA prepared by siRNA expression cassettes (SECs) against HPV18 E6 gene could significantly inhibit E6 gene expression and induce HeLa cells to apoptosis. The microarray analysis identified 359 differentially expressed genes, containing 307 up-regulated and 52 down-regulated genes. We analyzed the gene functions and cellular pathways in detail, including cell cycle related genes CCNG1, p21; apoptosis related genes CASP4, CASP6, IGFBP3, DFFA; ubiquitin proteolysis pathway related genes UBE3A, UBE2C; keratinocyte differentiation related genes KRT4, KRT6E, KRT18 and anti-oncogene RECK, VEL. And it can be concluded that cellular apoptosis induced by HPV18 E6-siRNA mainly depends on the P53 and ubiquitin proteolysis pathway to regulate the genes expression, consequently inhibiting the cell proliferation and promoting cell apoptosis. Meanwhile, the activation of anti-oncogene and upper regulation of immuniaztion related genes signified the degression of malignant extent of tumor cells after E6 inhibition.