Figure 2
Effect of pharmacological inhibition of constitutively active STAT3 by curcumin or phospho-STAT3 (Y705)-specific inhibitor stattic on miR-21 expression. SiHa cells (5 × 105 cells/w) seeded overnight were treated with indicated concentrations of curcumin or stattic for 24 h. Cellular proteins of the treated cells were examined for pSTAT3 (Y705), STAT3 and β-actin by immunoblotting or their RNA was checked for level of miR-21 and U6. Cells were treated in parallel in a 96 well plate to evaluate effect of treatment on cell viability by MTT assay. A &E. Graph showing cell viability by MTT assay following treatment with indicated doses of curcumin (A) or stattic (E). Values are mean ± SD of triplicate cultures with respect to untreated control. *p value <0.05. B &F. Dose-dependent effect of curcumin (B) or stattic (F) on levels of pSTAT3 (Y705). Cellular proteins (50 μg/lane) isolated from SiHa cells were examined first for pSTAT3 expression by immunoblotting, following which the blots were stripped and re-probed sequentially with STAT3 and β-actin antibodies as control. C, D, G &H. Inhibition of pSTAT3 levels accompanied loss of cellular miR-21 levels. Total miRNA pool isolated from SiHa cells treated with curcumin (C) or stattic (G) were examined for miR-21 by qRT-PCR. Expression of U6 similarly amplified in parallel was used as input control. Specific pSTAT3 and 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 pSTAT3, total STAT3 and miR-21 after inhibition of STAT3 phosphorylation in three independent experiments (D & H). *p value <0.05 compared to untreated cells.