Figure 7.

miR-21 regulates expression of PTEN and downstream kinases. (A) Real-time PCR for PTEN expression was assessed in RNA obtained from 11 HCC tumors (gray bars) and matching nontumoral sections (□). The normalized ratio of PTEN expression is indicated above each bar. (B) Cell lysates were obtained from normal human hepatocytes and HCC cell lines cultured in 100-mm culture dishes. Immunoblot analysis was performed for PTEN and for FAK activation using phosphorylation-state dependent antibodies. The blots were stripped and reprobed for α-tubulin as a loading control and for quantitation. Representative immunoblots are shown along with quantitative data showing the mean ± standard error from 4 separate blots. *P < .05 relative to expression in normal hepatocytes. (C) Normal human hepatocytes were transfected with 30 nmol/L miR-21 precursor or control. Immunocytochemistry for PTEN and phosphorylated FAK was performed after 72 hours. A decrease in PTEN expression along with an increase in FAK phosphorylation is observed in cells transfected with miR-21 precursor compared with controls. (D) HCC cells were transfected with 30 nmol/L anti–miR-21 or control inhibitor. Cell lysates were obtained after 72 hours for immunoblot analysis of PTEN protein expression and phosphorylation of its downstream target kinases Akt and FAK. Representative immunoblots and quantitative data (mean ± standard error) from 4 separate blots are shown. *P < .05 relative to expression in controls.