The Effect Of Proliferation, Apoptosis And Cell Cycle Onset In Human Cervical Cancer Cells Line Induced By HPV16 E6 Small Interfering RNA

Background & Objective: RNA interference is a new technology which blocks gene expression at the post-transcriptional, and a process in which gene silencing mechnism induced by double-stranded RNA (dsRNA) on molecular level. At the present, it has been developed in the field of virus-induced disease. Importantly, it has a huge application foreground in studying the gene function and gene therapy. This study is aimed to investigate if human papillomavirus 16 E6-specific small interfering RNAs (siRNAs) can be emplyed to inhibite the growth of cervical cancer cell line and the associated mechanism in vitro and in vivo. By doing so, we try to provide a new rationale for specific gene-therapy approach against cervical cancer. At the same time, may lay a theory foundation before applying to clinic.Method: 1. RNAi was induced using synthetic small interfering RNAs (siRNAs), which was transfected into CaSki cells line positive for human papillomavirus type 16 by lipofectamine. Cell growth curves , live cell ratioand the inhibition ratio of cells were measured by using cell counting. Amersco staining(AO), electron microscope and flow cytometry (FCM) were performed to evaluate the apoptosis induction of HPV16 E6 siRNA. The distributions of cell cycle were analyzed with the help of FCM at various time points of post-transfection. 2. At various time points of post-transfection, the expression level of HPV16 E6, p53, p21 mRNA were detected by using real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR), and the level of these proteins and CDK2 were measured by FCM. 3. To establish the nude mice model of cervical transplanted tumor, siRNA was distributed into the tumor. Inhibitory effect of HPV16 E6 siRNA on tumors was tested by measured the volume and the weight of tumors. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling(TUNEL) was performed to study the apoptosis in the tumor in response to targeting E6 mRNA. The expressions of E6 and p53 in tumor were measured by immunohistochemistry. In order to initially find out if siRNA is toxic to liver, the contents of alanine aminotransferase(ALT), aspartate aminotransferase(AST) were measured , and hematoxylin-eosin (HE) staining of liver and kidney were performed.Results: 1. The results showed that growth inhibition of E6 siRNA was demonstrated and livingness of cells was declined, the highest inhibition ratio was 77.4% at the 5th day after treating E6 siRNA. The characteristic morphological changes of apoptosis were observed by AO staining and electron microscope at 48 hour post-transfection, respectively. It had been showed that the highest cell apoptosis percentage was 40.5% at 48 hour post-transfection, the apoptosis index was 39.45% ± 1.48%, which evidentlyincreased compared to control (p<0.05). No substantial Gi arrest was observed by FCM analysis. 2. At 24 hour post-transfection , the level of E6 mRNA in response to E6-specific siRNA was decreased by 12.73 folds compared with control (p<0.05). However, cellular p53 and p21 mRNA levels appeared unaffected. 48 hour after transfection, the expression level of E6 protein was efficiently decreased, p53 and p21 protein level increased correspondly but cyclin dependent kinase 2 (CDK2) decreased . 3. Estibalished the nude mice model, the inhibition effect on tumors was demounstrated after treating with E6 siRNA by intratumor, the volume and weight of tumors were decreased (p<0.05). TUNEL assay demounstrated that apoptosis changes occurred more frequently than control after treating with E6 siRNA. The expression of E6 and p53 in tumor were negative. Toxic to liver had been no detected based upon the results that the contents of ALT and AST underwent no significant changes, and the histopathology of liver and kidney had no abnormal changes.Conclutions: It is identifided that HPV16 E6 siRNA could inhibite proliferation and induce apoptosis in cervical carcinoma cell lines CaSki in vitro and in vivo. Its inhibitory effect maybe due to specially and efficiently silence E6 mRNA expression, decrease the degradation of p53 protein, then recover the function activity of p53 protein. Thus, RNA interference techenology should provide a new rationale for specific gene-therapy approaches against HPV infection-related disease.