RBM10 inhibits cell proliferation of lung adenocarcinoma via RAP1/AKT/CREB signalling pathway

Abstract

Initial functional studies have demonstrated that RNA-binding motif protein 10 (RBM10) can promote apoptosis and suppress cell proliferation; however, the results of several studies suggest a tumour-promoting role for RBM10. Herein, we assessed the involvement of RBM10 in lung adenocarcinoma cell proliferation and explored the potential molecular mechanism. We found that, both in vitro and in vivo, RBM10 overexpression suppresses lung adenocarcinoma cell proliferation, while its knockdown enhances cell proliferation. Using complementary DNA microarray analysis, we previously found that RBM10 overexpression induces significant down-regulation of RAP1A expression. In this study, we have confirmed that RBM10 decreases the activation of RAP1 and found that EPAC stimulation and inhibition can abolish the effects of RBM10 knockdown and overexpression, respectively, and regulate cell growth. This effect of RBM10 on proliferation was independent of the MAPK/ERK and P38/MAPK signalling pathways. We found that RBM10 reduces the phosphorylation of CREB via the AKT signalling pathway, suggesting that RBM10 exhibits its effect on lung adenocarcinoma cell proliferation via the RAP1/AKT/CREB signalling pathway.

Keywords: AKT; CREB; RAP1; RNA-binding motif protein 10; lung cancer; proliferation.

Figure 1

RBM10 inhibits proliferation of A549 and H1299 cells. A, A549 and H1299 cells were transfected with lentiviruses expressing GV358‐RBM10 (RBM10) and GV248‐RBM10‐RNAi (RNAi) and with their corresponding controls, GV358 (NC) and GV248 (Vector), respectively. Total protein extracts from the cells were analysed by Western blotting for RBM10 expression. B, The effect of RBM10 on cell viability was measured by the CCK‐8 assay. C, Stably transfected cells were subjected to colony formation assays and incubated for 14 d. Colonies were photographed and counted. **P < 0.01, ***P < 0.001, ****P < 0.0001. Data represent mean values ± SD

Figure 2

RBM10 decreases the activation of RAP1. A, A549 cells were transfected with GV358‐RBM10 and with its corresponding control vectors (NC) and subjected to pull‐down assay using GST‐RBD, followed by immunoblotting with antibodies against RAP1; GV358‐RBM10 and NC were treated with PBS (P) or 100 μmol/L of the EPAC stimulator 8‐pCPT‐2’‐O‐Me‐cAMP (8‐pCPT) for 30 min, and GTP‐RAP1 was detected by western blotting. B: The effect of RBM10 overexpression on cell viability was measured by the CCK‐8 assay. C: A549 cells were transfected with GV248‐RBM10‐RNAi and with its corresponding control vector (V) subjected to pull‐down assay using GST‐RBD followed by immunoblotting with antibodies to RAP1; GV248‐RBM10‐RNAi and Vector were treated with DMSO (D) or 10 μmol/L of the EPAC inhibitor ESI‐09 for 30 min, and GTP‐RAP1 was detected by Western blotting. D: The effect of RBM10 knockdown on cell viability was measured by the CCK‐8 assay. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent mean values ± SD

Figure 3

Effect of RBM10 on the MAPK/ERK, P38 MAPK and AKT/CREB signalling pathways. A, The effect of RBM10 on ERK and P38 distribution in A549 cells was examined by western blotting. B, The effect of GV358‐RBM10 and treatment with the EPAC stimulator 8‐pCPT‐2’‐O‐Me‐cAMP on phospho‐AKT and phospho‐CREB distribution in A549 cells was examined by western blotting. C: The effect of GV248‐RBM10‐RNAi and treatment with the EPAC inhibitor ESI‐09 on phospho‐AKT and phospho‐CREB distribution in A549 cells was examined by Western blotting. D: A model for the role of RBM10 in lung adenocarcinoma cell proliferation. RBM10 inhibits proliferation via the RAP1/AKT/CREB signalling pathway. *P < 0.05, **P < 0.01. Data represent mean values ± SD

Figure 4

RBM10 inhibits lung tumour growth in vivo. A, A549 cells overexpressing RBM10 were subcutaneously injected into nude mice, and the mice were monitored for 27 d. A representative picture of tumours thus formed is shown. B, A growth curve of tumour volumes was constructed every 3 d for 27 d. C, Tumour weight was measured. D, A549 cells expressing RBM10‐RNAi were subcutaneously injected into nude mice, and the mice were monitored for 27 d. A representative picture of tumours thus formed is shown. E, A growth curve of tumour volumes was constructed every 3 d for 27 d. F, Tumour weight was measured. G, Photograph shows the dissected tumours formed by the inoculated infectants. H, Western blotting analyses of the expression of phospho‐AKT and phospho‐CREB in tumour tissues. **P < 0.01. Data represent mean values ± SD