RESUMEN
In the present research, we assumed that reducing the amounts of E6 and E7 oncoproteins by a specific siRNA sequence and recovering p53 and RB proteins, along with the recovery of the FOXO1 protein by applying anti-miR-182, would increase apoptosis and reduce proliferation rate in cancer cells. The HPV16-positive CaSki cervical cancer cell line was used. 48 hours after transfection of siRNA for targeting E6 and E7 oncoproteins and anti-miR-182, expression of its cellular targets p53, p21 and FOXO1 was assessed by real-time PCR, western blot analysis and immunocytofluorescence staining. In all treatments, apoptosis rate and viability were evaluated using Annexin-V-FITC apoptosis detection kits and MTT assays, respectively. Among the designed siRNAs, E6-1 and E7-2 proved the most effective in reducing E6 and E7 expressions by increasing the apoptotic rates to 12.4% and 16%, respectively, after 48 hours. Also, using anti-miR-182 increased apoptotic rate to 12.7% 48 hours after transfection of cervical cancer cells. The combinational use of either E6-1 or E7-2 siRNAs with anti-miR-182 resulted in a rise in apoptosis to 19.3% and 26%, respectively, higher than those obtained from the individual application of either without anti-miR-182. The simultaneous use of siRNA E6-1 and siRNA E7-2 with cisplatin increased sensitivity to cisplatin and reduced the viability of the cancer cells as compared to the use of cisplatin alone. The simultaneous use of cisplatin and anti-miR-182 had no considerable effect on viability or apoptosis rate compared to cisplatin alone.
Asunto(s)
Apoptosis/genética , Papillomavirus Humano 16/fisiología , MicroARNs/genética , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Cisplatino/farmacología , Femenino , Proteína Forkhead Box O1/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Oncogénicas Virales/deficiencia , Proteínas E7 de Papillomavirus/deficiencia , Interferencia de ARN , Proteínas Represoras/deficienciaRESUMEN
OBJECTIVE: To observe the effect of HPV16E7 specific expression vector on cell proliferation in cervical carcinoma SiHa cells. METHODS: The HPV16E7 siRNA expression vector and empty expression vector were transfected into SiHa cells by liposome. The effects on E7 mRNA and E7 protein expression, cell cycle phase and cell growth rate were examined respectively by real-time RT-PCR, FCM and MTT assay. RESULTS: The HPV16E7 siRNA expression vector significantly inhibited the expression levels of E7 mRNA and E7 protein, the inhibition rates being 92.15% and 84.30% respectively. It also inhibited the transition from G, phase to S phase and the growth of SiHa cell line. CONCLUSION: HPV16E7 specific siRNA expression vector could specifically and efficiently inhibit the expression of E7 gene and hence it could regulate cell cycle and inhibit cell proliferation in cervical carcinoma SiHa cells. siRNA expression vector
Asunto(s)
Vectores Genéticos/genética , ARN Interferente Pequeño/genética , Neoplasias del Cuello Uterino/patología , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas E7 de Papillomavirus/deficiencia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias del Cuello Uterino/genéticaRESUMEN
OBJECTIVE: To test of the effect of vector-based RNA interference (RNAi) technique on inhibiting HPV16E7 gene in CaSki cells of cervical cancer. METHODS: The HPV16E7-specific siRNA expression vectors P1, P2 and P3 were constructed and transfected into CaSki cells by liposome. The expression of HPV16E7 mRNA and protein were detected by real-time RT-PCR and Western blot. RESULTS: The expression of HPV16E7 mRNA and protein decreased with the transfection of P1, P2 and P3. Vector P1 had the strongest inhibition effect, with an inhibition rates of 92.86% and 81.0% for the expression of HPV16E7 mRNA and protein respectively three weeks after transfection. CONCLUSION: The expression of E7 gene in CaSki cells can be inhibited by HPV16E7-specific siRNA expression vector.