EZH2 silencing with RNA interference induces G2/M arrest in human lung cancer cells in vitro

Abstract

Nonsmall-cell lung cancer has a high mortality rate and poor prognosis. In the present study, we silenced EZH2 and explored the consequent cell cycle changes. The expression of cell-cycle-related proteins, including p53, p21, Cdc2, and cyclin B1, was detected with western blotting, and the cell cycle distribution was determined with flow cytometry. Inhibition of EZH2 expression changed the cell cycle distribution, in particular inducing G2/M arrest. Expression of Cdc2 and cyclin B1 was significantly decreased in A549 and HTB-56 cells after EZH2-siRNA treatment. In addition, p53 expression was increased by 21% and 18%, and p21 expression was increased by 31% and 23%, in A549 and HTB-56 cells, respectively, in the presence of EZH2-siRNA. This study clearly demonstrates that modulation of EZH2 expression with siRNA affects the cell cycle and the expression levels of p53 and p21, thereby changing cyclin B1 and Cdc2 expression and inducing G2/M arrest. These results may explain the observed antitumor activity of EZH2 silencing. Such explorations of the molecular mechanism of EZH2 will help us develop novel approaches to the diagnosis, treatment, and prevention of nonsmall-cell lung cancer.

Figure 1

Influence of EZH2-siRNA on the cell cycle. (a) EZH2 protein expression was analyzed with western blotting after transfection with EZH2-siRNA for 48 or 72 h. (b) The transfection efficiency of EZH2-siRNA was observed after 48 or 72 h. (c) The changes in the cell cycle were observed with FACS analysis after transfection with EZH2-siRNA for 72 h. (d) The effect of EZH2-siRNA after synchronizing the cell cycles at G₂/M phase using nocodazole. (e) After treatment with EZH2-siRNA, the expression of cell-cycle-related proteins, including Cdc2 and cyclin B1, was detected using western blotting. Each experiment was performed in triplicate.

Figure 2

Effect of EZH2-siRNA on p53 and p21 protein expression. p53 expression (a) and p21 expression (b) were strongly increased in both A549 and HTB-56 cells after transfection with EZH2-siRNA. (c) Relative expression of p53 mRNA was detected in the presence of GSK 126 or EZH2-siRNA. Each experiment was performed in triplicate.

Figure 3

Effect of EZH2-siRNA on cell cycle progression via the p53 and p21 pathway. (a) The effects of p53-siRNA on the expression of downstream molecular were observed by western blotting. (b) p53 and p21 expression was detected after pifithrin-α application. (c) and (d) Cdc2 and cyclin B1 expression were detected with western blotting after incubation with a p53 inhibitor (pifithrin-α) (c) or p21 inhibitor (adaphostin) (d) in the presence of EZH2-siRNA. (e) The changes in cell cycle progression were observed with FACS analysis after incubation with the p53 or p21 inhibitor in the presence of EZH2-siRNA. Each experiment was performed in triplicate.