Figure 5
Effect of targeting HPV16 E6 oncogene on miRs, Let-7a and miR-21 and level of active STAT3. SiHa cells transiently-transfected with indicated concentrations of HPV16 E6 -specific siRNA for 48 h were examined for viability, expression of Let-7a and miR-21. Scrambled siRNA (Scrl) was used as control. A. Graph showing cell viability by MTT assay following transient transfection with E6 siRNA at indicated doses. Values are mean ± SD of triplicate cultures with respect to untreated control. *p value <0.05. B. Dose-dependent effect of E6-siRNA on endogenous HPV16 E6 levels. Cellular proteins isolated from transfected SiHa cells were examined for HPV16 E6 protein expression by immunoblotting. Blots were stripped and re-probed with β-actin antibody as loading control. C &D. Targeting of E6 oncoprotein results in elevation of Let-7a levels and corresponding decline in miR-21.Total miRNA pools isolated from SiHa cells treated with E6-siRNA were examined for Let-7a (upper panel) and miR-21 (middle panel) by qRT-PCR (C). Expression of U6 similarly amplified in parallel was used as input control (lower panel). M: ΦX 174 HaeIII-digested molecular weight markers; UT-untreated cells. Specific HPV16 E6 bands were evaluated densitometrically and normalized against untreated control (UT). Let-7a and miR-21 fold change were calculated with respect to control by 2-ΔΔCt method. Graph showing mean ± SD of the fold change in expression of HPV16 E6, Let-7a and miR-21 after transient transfection of E6 in three independent experiments (D). *p value <0.05 compared to untreated cells. E. Effect of targeting HPV E6-siRNA on pSTAT3, STAT3 and PTEN expression levels. Cellular proteins (50 μg/lane) isolated from transfected SiHa cells were examined for expression of indicated proteins by immunoblotting. Blots were stripped and re-probed with β-actin antibody as loading control.