Functional role of RBM10 in lung adenocarcinoma proliferation

Abstract

Lung cancer is one of the most common causes of morbidity and mortality among malignant tumors worldwide. The poor prognosis of patients with lung adenocarcinomas is primarily due to its strong ability to invade and metastasize. Recent research has indicated that RNA‑binding protein 10 (RBM10) is mutated in lung adenocarcinoma, and is closely associated with tumor proliferation and apoptosis; however, the precise role of RBM10 in lung adenocarcinoma remains unclear. Our preliminary experiments (unpublished data) revealed that RBM10 expression was upregulated in lung adenocarcinoma cell lines and tissues. In this study, we first detected the protein expression level of RBM10 in lung adenocarcinoma cells and tissues, and we then examined the effects of RBM10 overexpression and downregulation (via small interfering RNA) on the proliferation and apoptosis of stable lung adenocarcinoma cells, along with its possible mechanisms of action. We also used clinical samples of lung adenocarcinomas to verify our results. We found that RBM10 protein was overexpressed in lung adenocarcinoma cells and tissues, and it reduced p53 expression (as detected by immunofluorescence assay and western blot analysis) in A549 cells and inhibited apoptosis (as shown by flow cytometric assay). RBM10 also promoted cell growth and proliferation in vitro and increased cell migration in a cell wound scratch assay. Furthermore, we found that RBM10 activated key proliferative signaling pathways [such as the epidermal growth factor receptor (EGFR), mitogen‑activated protein kinase (MAPK) and phosphoinositide 3‑kinase (PI3K)‑AKT pathways] and inhibited apoptotic pathways. In addition, we demonstrated that a high expression of RBM10 protein in patient tissue samples was associated with a shorter overall survival time and a poor prognosis. On the whole, the findings of this study indicate that RBM10 may function as an oncogene in lung cancer, and may thus prove to be a novel therapeutic target for the prophylaxis and treatment of lung adenocarcinomas.

Keywords: lung adenocarcinoma; RNA-binding protein 10; proliferation; apoptosis.

Figure 1

Protein expression of RBM10 in cells and tissues. (A) Protein expression of RBM10 examined by immunofluorescence assay; magnification, ×400 and quantitative analysis of immunofluorescence assay (^*P<0.01, A549 cells vs. the HLF cells and ^#P<0.01, H1299 cells vs. the HLF cells. (B) Expression of RBM10 examined immunohistochemistry in tumor tissues and NCTs (n=6); magnification, ×400. (C) Expression of RBM10 in tumor tissues and NCTs examined by western blot analysis and quantitative analysis; ^#P<0.05 vs. NCT. NCT, (adjacent) non-cancerous tissues; HLF cells, human lung fibroblast cells.

Figure 2

Effects of RBM10 protein on the apoptosis of tumor cells. (A) Effects of the downregulation or upregulation of RBM10 protein on p53 protein expression examined by immunofluorescence assay in A549 cells; magnification, ×400. (B) Effects of the downregulation or upregulation of RBM10 on p53 expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. ^#P<0.05, RBM10 pcDNA3.1 group compared with the control or mock group and ^*P<0.05, RBM10 siRNA group compared with the control or mock group. (C) The level of cell apoptosis following the knockdown of RBM10 protein, examined by flow cytometry and the apoptotic rate in A549 cells and H1299 cells. ,^#P<0.05 vs. control or RBM10 siRNA (−) group.

Figure 3

Evaluation of the proliferative ability of lung adenocarcinoma cells following the knockdown of RBM10 expression examined by cell colony formation assay. (A) In A549 cells, western blot analysis was used to verify the successful transfection of different DNA sequence cells and the cell proliferation on soft agar. (B) In H1299 cells, western blot analysis was used to verify the successful transfection of different DNA sequence cells and the cell proliferation on soft agar.

Figure 4

Effect of the knockdown of RBM10 expression on H1299 cell migration examined by cell wound scratch assay. Verification of decreased RBM10 levels after silencing by shRNA in H1299 cells by western blot analysis and cell migration compared with the control and vector control (PLKO.1) examined by cell wound scratch assays at 0, 12 and 24 h.

Figure 5

Effects of the upregulation or downregulation of RBM10 expression on EGFR expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. ^#P<0.05, RBM10 pcDNA3.1 group vs. control or mock group and ^##P<0.05, RBM10 siRNA group vs. control or mock group.

Figure 6

Effects of RBM10 protein levels on proliferative signaling pathways. Expression levels of key proteins in MAPK and PI3K signaling pathways following the upregulation or downregulation of RBM10 protein expression examined by western blot analysis and quantitative analysis of relative protein expression in A549 cells and H1299 cells. ^*P<0.05 and ^**P<0.01, compared with control or mock groups.

Figure 7

Effects of the upregulation or downregulation of RBM10 expression on apoptotic proteins (Bax, Bcl-2 and caspase-8). (A) Effects of RBM10 on apoptotic proteins examined by western blot analysis and quantitative analysis of relative protein expression in A549 cells. (B) Effect of RBM10 on apoptotic proteins examined by western blot analysis and quantitative analysis of relative protein expression in H1299 cells. P<0.05, compared with the control or mock group (^*Bax protein, ^#Bcl-2 protein, ^##caspase-8 protein).

Figure 8

Associations of the different protein expression levels of RBM10 with the clinical prognosis of patients with lung adenocarcinoma. (A) Kaplan-Meier analysis of overall survival of patients with lung adenocarcinoma with different levels of RBM10 expression in the nucleus (P<0.05, log-rank test). (B) Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients with different levels of RBM10 expression in the cytoplasm (P<0.05, log-rank test).