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目的以RNA干扰技术为手段,HPV编码的癌蛋白E6为靶标,探讨靶向HPV16-E6的siRNA对宫颈癌细胞生物学行为的影响,并试图阐明该实验的临床意义。方法构建靶向HPV16-E6的siRNA表达载体,应用体外转染试剂转染HPV16-E6阳性的宫颈癌CaSki细胞。以RT-PCR检测CaSki细胞中E6蛋白的mRNA的表达,借助细胞色素c测定来分析细胞凋亡相关分子的表达和活性,从而研究靶向HPV16-E6的siRNA诱导细胞凋亡的分子机制。结果RT-PCR检测结果表明,将靶向HPV16-E6的siRNA的表达载体瞬时转染到HPV16-E6阳性的CaSki细胞后,其所含E6蛋白质和mRNA的表达下调;Western blotting检出抑凋亡蛋白Bcl-2的表达亦告下调;细胞色素c释放实验结果显示,它从线粒体释放到细胞浆中,从而诱导细胞凋亡。结论靶向HPV16-E6的siRNA能够有效抑制细胞增殖并诱导细胞凋亡。它为研究重要致瘤蛋白HPV16-E的功能开辟了新途径,给HPV16-E6阳性肿瘤的靶向基因治疗提供新的实验依据,并探索了HPV感染及宫颈癌的新基因疗法。
Objective To investigate the effect of siRNA targeting HPV16-E6 on the biological behavior of cervical cancer cells by means of RNA interference technology and HPV-encoded oncoprotein E6, and to elucidate the clinical significance of this experiment. Methods siRNA targeting HPV16-E6 expression vector was constructed and transfected into HPV16-E6 positive cervical cancer CaSki cells using in vitro transfection reagent. The expression of E6 protein mRNA in CaSki cells was detected by RT-PCR, and the expression and activity of apoptosis-related molecules were analyzed by cytochrome c assay to investigate the molecular mechanism of siRNA-induced apoptosis in HPV16-E6 cells. Results The results of RT-PCR showed that the expression of E6 protein and mRNA in HPV16-E6-positive CaSki cells was transiently transfected with siRNA targeting HPV16-E6. The expression of E6 protein and mRNA was down-regulated by Western blotting The expression of Bcl-2 protein was also down-regulated. The cytochrome c release assay showed that it was released from the mitochondria into the cytoplasm to induce apoptosis. Conclusion siRNA targeting HPV16-E6 can effectively inhibit cell proliferation and induce apoptosis. It opens up new avenues for studying the function of HPV16-E, an important oncogenic protein, providing new experimental evidences for targeted gene therapy of HPV16-E6 positive tumors and exploring new gene therapy for HPV infection and cervical cancer.