Applications Of The Plasmid-mediated Antisense Approach For Gene Therapy In Cervical Cancer

PART I Antisense targeting human papillomavirus type 16 E6 andE7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cellsObjective: Human papillomavirus type 16 (HPV-16) is a high-risk DNA tumour virus involved in the development of cervical carcinomas. Substantial studies have demonstrated that E6 and E7 oncoproteins of HPV-16 could induce cell proliferation and immortalization. Repression of E6 and/or E7 oncogenes may induce cervical cancer cells to undergo apoptosis or senescence. The purpose of this study was to determine whether activation of the p53 and retinoblastoma (Rb) pathway by HPV-16 E6 and E7 repression was responsible for apoptosis and senescence of cervical cancer cells and to explore the potential of an antisense RNA (AS) transcript for gene therapy of cervical cancer.Method: The antisense RNA directed against HPV-16 E6 and E7 (16AS) was constructed, and its effects on cell apoptosis and senescence of SiHa cervical carcinoma cells harboring HPV-16 were analyzed. The efficiency of 16AS was evaluated with RT-PCR, Western Blotting, flow cytometry analysis, Hoechst 33258 staining, senescent cell morphology observation and senescence associatedβ-galactosidase staining.Results: The sufficient repression of HPV-16 E6 and E7 oncogenes were achieved in 16AS-transfected SiHa cells, which led to obvious apoptosis and replicative senescence of tumor cells. Furthermore, the downregulation of HPV16 E6 and E7 by 16AS transfection resulted in remarkable increase of both p53 expression and hypophosphorylated p105Rb level in SiHa cells.Conclusion: These results demonstrate that reduction of E6 and E7 expression is sufficient to induce SiHa cells to undergo apoptosis and senescence, and suggest that transfection of cervical cancer cells with HPV-16 E6 and E7 antisense RNA is a potential approach to treat HPV-16 positive cervical cancers. PART II Antisense targeting to human papillomavirus (HPV) 18 E6E7 affects the proliferation and apoptosis of human cervical carcinoma HeLa cellsObjective: The eukaryotic fluorescent expression vector carrying antisense human papillomavirus (HPV) 18 E6E7 was constructed and transfected into human cervical carcinoma HeLa cells to investigate its effect on the growth and prolifection of HeLa cells.Methods: The HPV18 E6E7 716bp was amplified by PCR and then the PCR product was inversely inserted into pEGFP and contructed the recombinant eukaryotic expression plasmid pEGFP-HPV18E6E7as (EGFP-18AS). The recombinant was further transfected into HeLa cells. RT-PCR and western blot were respectively used to detect the mRNA or protein expression of HPV18 E6/E7 in the HeLa cells. The MTT method was performed to dynamically monitor the survived cells and the cell apoptosis was observed by flow cytometer and fluorescence microscope.Results: The protein and mRNA expression levels of HPV18 E6/E7 in HeLa cells transfected with EGFP-18AS (HeLa/18AS) were both lower than those of the control groups including the HeLa cells transfected with EGFP (HeLa/EGFP) and untreated HeLa cells. The numbers of survived cells in HeLa/18AS cells became significantly lower than those of the control (all P<0.05). The rate of sub-G1 phase was also revealed, the apoptotic rate in HeLa/18AS cells was 47.21%, significantly higher than those of the HeLa/EGFP and untread HeLa cell (14.18% and 3.36% respectively, both P<0.05). Furthermore, an increased number of cells with chromosome condensation and fragmentation were found in HeLa/18AS cells as compared with HeLa/EGFP cells.Conclusion: The recombinant pEGFP-HPV18E6E7as can effectively inhibit the growth and proliferation of human cervical carcinoma HeLa cells, and further induce the cell apoptosis. The antisense RNA technology was proved to be available, and it might provide a new way to gene therapy of the cervical carcinoma. PART III RNA interference against HPV16 E7 oncogene leads to apoptosis in SiHa cervical carcinoma cellsObjective: Human papillomavirus type 16 (HPV16), a causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a Small interfering RNA (siRNA) targeting HPV16 E7 region to degrade both E6, E6* and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. The shRNA that targets HPV16 E7 was tested in HPV6-positive cell lines to investigate its effect and investigate its mechanism of action.Method: Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. To examine the effects of pSIREN-16E7 on the expression of E6 and E7 oncogenes and on the cell growth of HPV16-related cervical cancer cells, we applied RT-PCR, Western Blotting, MTT assay, the Annexin V apoptosis assay and flow cytometry in our study. Results: Using human papillomavirus (HPV) 16-transformed cells as a model system, Our results indicated selective degradation of E6 and E7 mRNAs. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. HPV-negative cells appeared unaffected by the anti-viral siRNAs.Conclusion: we demonstrated for the first time that HPV16 E7-specific targeting can induce simultaneous E6 and E7 suppression. Therefore, RNAi using E7 shRNA may have potential as a gene-specific therapy for HPV16-related cancers.