Effect of targeting STAT3 expression by RNA interference on miR-21 expression. SiHa cells (2 × 105 cells) transiently-transfected with indicated concentrations of STAT3-specific siRNA for 48 h were examined for viability, STAT3 protein and transcript levels and expression of miR-21. Scrambled siRNA (Scrl) was used as control. A. Graph showing SiHa cell viability by MTT assay following transient transfection at indicated doses of STAT3 siRNA. Values are mean ± SD of triplicate cultures with respect to untreated control. *p value <0.05. B &C. Dose-dependent effect of STAT3-siRNA on STAT3 expression. Cellular proteins (50 μg/lane) isolated from transfected SiHa cells were examined for STAT3 protein expression by immunoblotting (B). Blots were stripped and re-probed with β-actin antibody as loading control. (C) Representative Ethidium bromide-stained agarose gel (3%) photograph showing levels of STAT3 transcripts measured by RT-PCR (upper panel) in cDNA derived from STAT3 siRNA-treated SiHa cervical cells. GAPDH RT-PCR was used as internal control for input RNA (lower panel). M: ΦX 174 HaeIII-digested molecular weight markers; UT-untreated cells. D &E. Inhibition of STAT3 levels accompanied loss of cellular miR-21 levels. Total miRNA pool isolated from treated SiHa cells was examined for levels of miR-21 by qRT-PCR as described in ‘Methods’. Level of ubiquitously-expressed microRNA U6 similarly amplified in parallel was used as input control of miRNA. (D). Specific STAT3 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 STAT3 protein, STAT3 transcripts and miR-21 after STAT3 inhibition in three independent experiments (E). *p value <0.05 compared to untreated cells.