Figure 3

Effect of miR-21 silencing on cell viability, on expression of PTEN and active STAT3 level. SiHa cells transiently-transfected with indicated concentrations of miR-21 – specific inhibitor for 48 h were examined for viability and expression of downstream target PTEN. A. Graph showing cell viability by MTT assay following transient transfection with miR-21 inhibitor at indicated doses. Values are mean ± SD of triplicate cultures with respect to untreated control. *p value <0.05. B. Representative gel photograph showing reduced levels of miR-21 in treated cells (upper panel). Total miRNA pool isolated from treated SiHa cells was examined for miR-21 by qRT-PCR. 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. C &D. Dose-dependent accumulation of PTEN in cells treated with miR-21 inhibitor. Cellular proteins isolated from treated SiHa cells were examined first for PTEN expression by immunoblotting, following which the blots were stripped and re-probed with β-actin antibody as loading control (C). Specific PTEN bands were evaluated densitometrically and normalized against untreated control (UT). miR-21 fold change was calculated with respect to control by 2^-ΔΔCt method. Graph showing mean ± SD of the fold change in expression of miR-21 and PTEN protein after inhibition of miR-21 in three independent experiments (D). *p value <0.05 compared to untreated cells. E. Concomitant reduction of pSTAT3 by miR-21 inhibitor. Cellular proteins isolated from treated SiHa cells were examined first for pSTAT3 and STAT3 expression by immunoblotting, following which the blots were stripped and re-probed with β-actin antibody as loading control.