PPM1D silencing by RNA interference inhibits the proliferation of lung cancer cells

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Abstract

Background: PPM1D (protein phosphatase, Mg2+/Mn2+ dependent, 1D) has been reported to be involved in multiple human tumors. This study was designed to investigate the functional role of PPM1D in lung cancer cells. Methods: Expression levels of PPM1D were analyzed in A549 and H1299 cells by real-time PCR and Western blotting. Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down PPM1D expression in both cell lines. The effects of PPM1D on lung cancer cell growth were investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), colony formation and flow cytometry assays. Results: Knockdown of PPM1D in lung cancer cells resulted in decreased cell proliferation and impaired colony formation ability. Moreover, flow cytometry analysis showed that knockdown of PPM1D arrested cell cycle at the G0/G1 phase. Furthermore, PPM1D silencing downregulated the expression of cyclin B1 in H1299 cells. Therefore, it is reasonable to speculate that the mechanisms by which PPM1D knockdown alleviates cell growth may be partly via the induction of cell cycle arrest due to the suppression of cyclin B1. Conclusions: These results suggest that PPM1D silencing by RNA interference (RNAi) may be a potential therapeutic approach for the treatment of lung cancer.

Figures

  • Figure 1 Expression levels of PPM1D in lung cancer cells. (A) Gene expression levels of PPM1D in A549 and H1299 cells analyzed by real-time PCR. (B) Protein expression levels of PPM1D in A549 and H1299 cells analyzed by Western blotting. (C) PPM1D gene expression levels in Lv-shPPM1D infected, Lv-shCon infected and non-infected (Con) A549 cells. (D) PPM1D gene expression levels in Lv-shPPM1D infected, Lv-shCon infected and non-infected H1299 cells. (E) Light microscopic and florescence microscopic pictures of Lv-shPPM1D infected, Lv-shCon infected and non-infected A549 and H1299 cells. Values are the mean of three independent experiments; bars represent SD. ***P < 0.001 in comparison with control. Con, control; GFP, green fluorescent protein.
  • Figure 2 Effect of siRNA-mediated PPM1D knockdown on the proliferation of lung cancer cells. A549 (A) and H1299 (B) cells analyzed using the MTT assay. (C). Light microscopic and fluorescence microscopic pictures of the colonies of A549 cells. Colonies were stained with Giemsa staining after 8 days of infection. Number of colonies of Lv-shPPM1D infected, Lv-shCon infected and non-infected A549 (D) and H1299 (E) cells. Values are the mean of three independent experiments; bars represent SD. **P < 0.01 and ***P < 0.001 in comparison with the control. Con, control; OD, optical density.
  • Figure 3 Fluorescence-activated cell sorting histograms showing cell cycle distributions of Lv-shPPM1D infected, Lv-shCon infected and non-infected H1299 cells. Con, control. Each line is the repeats form three independent experiments.
  • Figure 4 Effect of PPM1D knockdown on the cell cycle distribution of H1299 cells. (A) Percentage of cells residing in each checkpoint of the cell cycle. (B) Cyclin B1 protein expression levels of Lv-shPPM1D infected, Lv-shCon infected and non-infected H1299 cells analyzed by Western blotting. Values are the mean of three independent experiments; bars represent SD. *P < 0.05 and **P < 0.01 in comparison with the control. Con, control.

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Zhang, C., Chen, Y., Wang, M., Chen, X., Li, Y., Song, E., … Peng, H. (2014). PPM1D silencing by RNA interference inhibits the proliferation of lung cancer cells. World Journal of Surgical Oncology, 12(1). https://doi.org/10.1186/1477-7819-12-258

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