Effect of targeting STAT3 expression by RNA interference on the expression of p53 & pRb and E6 & E7 and on viability of cervical cancer cells.

<p>(A) Specific STAT3 siRNA inhibits STAT3 and pSTAT3 expression in cervical cancer cells. Cellular proteins (50 µg) isolated from SiHa cells (2×10⁵ cells) treated with the indicated concentrations of STAT3 and control siRNA (Scr) for 48 h were examined for pSTAT3 (Y705), STAT3, p53, or pRb by immunoblotting as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067849#s2" target="_blank">Methods</a>’. Blots were stripped and reprobed with β-actin as loading control. (B) Nuclear proteins (10 µg/lane) isolated from SiHa cells treated with STAT3 or control siRNA were incubated with radiolabeled STAT3 oligos to check STAT3-specific DNA binding activity by EMSA. NS- Non specific band. (C) Blocking STAT3 by specific siRNA abrogates expression of HPV16 oncogenes E6 and E7 in cervical cancer cells. Cellular proteins isolated from SiHa cells (2×10⁵ cells) treated with the STAT3 and control siRNA (160 nM) for 48 h were examined for expression of HPV16 oncogenes E6 and E7 by immunoblotting. Blots were stripped and reprobed with β-actin as loading control. (D) STAT3 inhibition was accompanied with reduced proliferation/ loss of cell viability in HPV16 positive cervical cancer cells. HPV16 positive SiHa cells and HPV negative C33a cells (2×10⁵ cells) treated with the indicated concentration of STAT3-specific and control siRNA for 48 h were harvested by trypsinization and counted for live cells using Trypan blue vital dye. Experiment was performed in triplicate, error bars indicates mean±SD. Lower panel indicates photomicrographs of respective control and treated SiHa and C33a cell cultures prior to their harvesting for cell counting.