The Effect Of Silencing HPV16/18E6/E7mRNA On The Malignant Biological Behavior In SiHa And HeLa

Objective: Human papillomavirus (HPV) oncogenes E6and E7play an important role incervical cancer. The E6/E7oncogenes show an important value in the cervical cancerscreening and treatment of HPV. However, the carcinogenic mechanism of E6/E7remainsunclear. In this study, we observed the malignant biological behavior in stable-transfectedcervical cancer cells, which include the expression of tumor suppressor proteins p53, pRband p16, cell proliferation, cell cycles, migration. In addition, we detected the cellapoptosis after treatment of Cisplatin and the amplification of hTERC gene. We aimed toexplore the role of E6/E7in the carcinogenesis and progression of cervical cancer and tosuppose molecular mechanism, and to assess the value of shRNA in HPV treatment andanti-tumor.Methods:1. shRNA plasmid construction: we designed6shRNA consequences accordingthe designed rules aimed at HPV16and18E6/E7mRNA respectively. Then plasmids weretransfected into SiHa and HeLa cells by Electrical-transfection. Stable-transfected silencedmomclonal cells expressing shRNA and negative control shRNA were selected for furtherexperiments.2. In vitro:①Using reverse-transcription PCR, the effects of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;②By real-time quantitative RT-PCR, theeffect of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;③ByWestern-blot, the expression of anti-tumor proteins p53, pRb, and p16in SiHa cell andHeLa cell were investigated;④The growth rate of HeLa cell and SiHa cell treated withshRNA was determined with MTT assay; Transwell assay be used to observe cellmigration in vitro; Cell cycles and apoptosis of SiHa cell HeLa cell after48h treated withcisplatin were observed with flow cytometer;⑤Amplication of hTERC gene wasdetected by FISH.3. In vivo: Nude mices were injected2×10~6the stable-transfected SiHa cell and HeLa cells, respectively.①Volumn of transplantation tumors were measured and compared;②Expression of hTERC gene on tumor tissue was detected by FISH.Results:1. Interference of shRNA: we successfully constructed6shRNA plasmids. Thesequence-specifical shRNA suppressed the HPV16/18E6/E7mRNA expression effectively.In SiHa cells, quantitative real-time PCR showed that E6and E7expression reduced byshRNA pSi-16-1, pSi-16-2, pSi-16-3were85.4%and54.9%；91.9%and63.2%；84.7%and71.1%, respectively. And in HeLa cells, the suppression ratios were91.3%and55.8%；90.4%and7.8%；87.2%and81.0%, respectively.2.In vitro:①pSi-shRNA can silence expression of HPV E6/E7mRNA effectively;②pSi-shRNA can increase the expression of p53, pRb and p16in SiHa cell and HeLa cell;③It was confirmed by MTT assay that pSi-shRNA can significantly inhibit theproliferation of SiHa cell and HeLa cell（P＜0.05);④After be treated by colchicine, thepropotions of cell in G0/G1stage increased. However, the propotions of cell in S stagedecreased;⑤By Transwell assay, cell migration ability were weaken;⑥After betreated by cisplatin for48h, apotosis rate were increase. Silencing of E6/E7increased SiHacell sensitivity to cisplatin;⑦Silencing of E6/E7inhibited expression of hTERC gene.The normal cell was increased and the cell signal of4:4was decreased.3. In vivo:①the growth of tumor was inhibited by silencing of E6/E7, the volume of thetumor in pSi-shRNA were lower than that of negative control (P＜0.01）;②the expressionof hTERC gene were dow-regulation by silencing of E6/E7.Conclusion: The recombinant plasmids can silence the expression of HPV16and HPV18E6/E7successfully. Silencing of HPV E6/E7resulted in the expression of p53、pRb、p16increase, and cell growth inhibition, migration ability weaken, and made the cell cycleasset in G0/G1stage in SiHa and HeLa cells. In addition, Silencing of HPV E6/E7increasedcell sensitivity to cisplatin. A closely relationship between the E6/E7and hTERC gene inSiHa and HeLa both in vitro and in vivo was found.