In-depth Study Of Chemically Synthesized Sirna Silence Siha Cell E6 Gene Expression

ObjectiveE6 and E7 oncoproteins play important roles in initiation and malignant progression of human cervical carcinoma, when the DNA of high-risk types of human papillomaviruses (HPV) integrates into chromosome of human cervical epithelial cells. E6 and E7 proteins modulate cellular cycle regulator factor P53 and Retinoblastoma protein (pRb) and play critical role in the generation and development of cervical cancer. Silencing these oncogenes is considered to be applicable in moleculer therapies of human cervical cancer. RNA interference (RNAi) may serve as a novel and powerful technology for the research of gene function and gene treatment. It can silence gene expressing by degrading mRNA specifically and efficiently, and it has been widely applied for the research in gene function of virus and gene treatment on virus associated tumors. For our study, siRNA specifically targeting of the HPV16 E6 oncogene was used to treat on SiHa, one HPV16-positive human cervical cancer cell, to evaluate the special inhibitory effects of E6 and E7 genes. And the expressions of proteins P53, pRb, VEGF-C and Survivin were also examined.Methods(1) We desined siRNA which specifically target of HPV16 E6 gene according to Tuschl's principle. The sequences were analyzed for homology by BLAST.(2) HPV16 E6 siRNA was transfected into SiHa cells by Lipofectamine 2000.(3) Total RNAs were extracted after post-transfection 24h, 48h and 72h. And the expression of E6 and E7 mRNA were detected by RT-PCR. Western Blot was used to detect the change of P53, pRb, Survivin and VEGF-C proteins in the SiHa cells of post-transfection 72h.Results(1) The specific HPV16 E6 siRNA decreased E6 mRNA expression to 34.5%, 37.3% and 40.5% post-transfected 24h, 48h and 72h as compared with Lipofectamine2000 control groups respectively (P<0.05). And the E7 mRNA expressions were decreased by 63.9%, 67.1%, and 58.3% as compared with Lipofectamine 2000 control groups (P<0.05).(2) The protein expressions of P53, pRb, VEGF-C and Survivin in SiHa cells transfected with E6 specific siRNA 72h were detected by Western Blot. Compared with Lipofectamine2000 control groups, P53 and pRb expressions increased by 108.4% and 76.5%, and VEGF-C were decreased by 28.7% (P<0.05). And the expression of Survivin protein in experiment group had no difference with control group (P>0.05).Conclusions (1) Our results indicated that this siRNA can induce simultaneous E6 and E7 suppression.(2) Cellular P53 and pRb were activated and the expression of VEGF-C was decreased via down-regulation the expressions of E6/E7 oncogenes. But the silencing effects had no change on the expression of protein Survivin.