The Experimental Study On The Treatment Of Cervical Cancer With The HPV16-E6-Targeted SiRNA

Objective To study the expression levels of HPV16-E6 in the cervical cancer tissues and evaluate the clinical significance of applying the immunohistochemical determination of HPV16-E6 to the early diagnosis of cervical cancer. To study the effect of the HPV16-E6-targeted siRNA on the biological behaviors of the cervical cancer cells by using RNA interference targeting the HPV-encoded oncoprotein HPV16-E6 so as to open up a new way for the functional study of this important oncoprotein and provide n ew bases theoretically and experimentally for the gene-targeted treatment of the HPV16-E6-positive diseases, especially HPV infection and cervical cancer.Method Pathological sections of cervical cancer tissues were collected and the HPV16-E6 expression levels in cervical cancer tissues were determined immunohistochemically; The expression vectors of siRNA targeting HPV16-E6 were constructed and transfected into the HPV16-E6-positive CaSki cells. Levels of the HPV16-E6 rnRNA and the mRNA encoded proteins expressed in the CaSki cells were tested by RT-PCR and Western blotting, respectively; The cell proliferation activities were tested by MTT analysis, and the cell cycle and apoptosis were detected with flow cytometry. Also, the expression levels and activities of the apoptosis-associated molecules were detected by Western blotting, cytochrome c determination and caspase activity analysis. The molecular mechanism of apoptosis induced by siRNA targeting HPV16-E6 was studied. The effect of siRNA targeting HPV16-E6 on the transplanted tumor in cervical cancer nude mice was observed in the cervical cancer nude mice models through multi-site injection of siRNA targeting HPV 16-E6 into the transplanted tumor.Results The immunohistochemical detection of the pathological section of cervical cancer tissues showed that the HPV16-E6 positive rates in chronic inflammation tissues, CINs and cervical cancer tissues were 8%, 22.7% and 55.9%, respectively. Among the cervical cancer tissues, the HPV16-E6 positive rates in stageâ… ,â…¡andâ…¢were 44.4%, 55.6% and 80%, respectively. After the expression vectors of siRNA targeting HPV16-E6 were transiently transfected into the HPV16-E6- positive CaSki cells, a variety of considerable changes were detected: The Western blotting and RT-PCR analyses showed that the expression of the E6 protein and mRNA were down-regulated; The MTT analysis showed that the cell proliferation was inhibited; The flow cytometry analysis showed that the cell cycle was blocked in the G₁/S phase and the cell apoptosis was increased. Further investigation also showed that the expression of the antiapoptosis protein Bcl-2 was down-regulated; The the cytochrome c liberation test showed the cytochrome c was released from mitochondria into cytoplasm, and hence the induction of cell apoptosis; The Caspase activity analysis also showed that Caspase 3, Caspase 8, and Caspase 9 in the cell were activated. The in vivo experiment demonstrated that the siRNA targeting HPV16-E6 could significantly inhibit the transplanted tumor in cervical cancer nude mice and induce cell apoptosis.Conclusion The expression level of HPV16-E6 is closely related to the occurrence and development of cervical cancer. HPV16-E6 positive rate increases with the exacerbation of the disease. The immunohistochemical detection of the expression levels of HPV16-E6 in the cervical cancer tissues is helpful to the early diagnosis of the disease. In the CaSki cells of cervical cancer, the HPV16-E6-targeted siRNA can inhibit the cell proliferation, induce a cell cycle blockage at G₀/G₁ phase and consequently the cell apoptosis. The possible mechanism of the cell apoptosis induced by the HPV16-E6-targeted siRNA may be deduced as this: With the inhibition of HPV16-E6 expression by the HPV16-E6-targeted siRNA, the expression of Bcl-2 is down-regulated; The permeability of the mitochondrial membrane is changed and the cytochrome c is released, resulting in a celll apoptosis. The inhibition of the HPV16-E6 leads to activation of Caspase3, Caspase8 and Caspase9, with a higer increase in activities of Caspase 3 and Caspase 9 than that of Caspase 8. These suggest that the HPV16-E6-targeted siRNA in the CaSki cells of cervical cancer can activate not only the mitochondrial pathway, but also the death receptor pathway of cell apoptosis, though the siRNA chiefly activates the mitochondrial pathway. In the cervical cancer nude mice models, the HPV16-E6-targeted siRNA significantly inhibits the transplanted tumor and induces cell apoptosis. So we believe that the HPV16-E6-targeted siRNA inhibits the proliferation of cervical cancer cells and induces the cell apoptosis, and the oncoprotein E6 encoded by the HPV gene may be regarded as a potential cadidate of important target molecule for gene therapy.