Inhibition of non-small-cell lung cancer cell proliferation by Pbx1

Abstract

Lung cancer is one of the most deadly human cancers and continues to be a major unsolved health problem worldwide. Here, we evaluate the function of Pbx1 in the proliferation of non-small-cell lung cancer (NSCLC). In contrast with its known proliferative function, we found that Pbx1 inhibits the proliferation of lung cancer cells. In particular, Pbx1-specific RNA interference resulted in increased proliferation in lung cancer cells. In addition, histone H3 phosphorylation was also increased following inhibition of Pbx1 expression. In contrast, Pbx1 overexpression repressed the proliferation of lung cancer cells and inhibited DNA synthesis. Collectively, our data indicate that Pbx1 inhibits proliferation in lung cancer cells, suggesting a complex role for Pbx1 in modulating the proliferation of cancer cells and making this protein a potential new target for lung cancer therapy.

Keywords: Pbx1; non-small-cell lung cancer (NSCLC); proliferation.

Figure 1

Efficiency of Pbx1 RNAi. (A) A549 cells were transfected with Pbx1 or scrambled RNAi for 72 h. mRNA levels were then analyzed (mean SD, n=3, ***P<0.001); (B) cells were treated as in (A) and subjected to western blotting analysis. Experiments were performed in triplicate and representative images are shown.

Figure 2

Pbx RNAi inhibits NSCLC cell proliferation. (A) A549 cells were transfected for 24 h and then colony formation assays were performed. Representative images are shown; (B) quantification of clones in (A) (mean SD, n=3, *P<0.05); (C) scrambled or Pbx1 RNAi constructs were transfected into A549 (left panel) and SPC-A1 (right panel) cells for 48 h. Cells were subjected to MTT analysis (mean SD, n=3, **P<0.01).

Figure 3

Pbx1 regulates cell cycle progression in NSCLC cells. (A) Cell cycle analysis was performed after RNAi transfection in A549 cells for 72 h. M1 indicates G0/G1 phase, M2 indicates S phase and M3 indicates M phase; (B) cells were transfected as in (A) and then fixed for immunofluorescence to detect histone H3 phosphorylation. Representative images from three parallel experiments are shown; (C) quantification of clones in (B) (mean SD, n=3, ***P<0.001).

Figure 4

(A) A549 cells were transfected for 72 h and then subjected to RT-PCR analysis; (B) A549 cells were transfected with RNAi for 72 h and then lysed for western blotting analysis. Specific antibodies were used as indicated. Experiments were performed in triplicate, and representative images are shown.

Figure 5

Pbx1 overexpression represses NSCLC cell proliferation. (A) Determination of Pbx1 protein levels by western blotting analysis. GAPDH was used as a loading control. Representative images from three independent experiments are shown; (B) MTT analysis of Pbx1-overexpressing A549 cells (mean SD, n=5, *P<0.05); (C) EdU was added to control or Pbx1-overexpressing A549 cells; cells were cultured for 8 h and then collected for EdU analysis (mean SD, n=5, *P<0.05).