Molecular Mechanism Of RBM10 Inhibiting Proliferation Of Non-small Cell Lung Cancer By Regulating RAP1/AKT/CREB Signal Transduction Pathway

Background:Lung cancer is the most commonly diagnosed cancer(11.6%)and the leading cause of cancer-caused death(18.4%).For purposes of treatment,lung cancer is classified either as small cell lung cancer(13%)or non-small cell lung cancer(NSCLC;87%),for which the overall 5-year survival rate is only 18.2%.In recent years,molecular targeted therapies have dramatically improved progression-free survival and overall survival for NSCLC patients with activated oncogenes such as mutant EGFR or translocated ALK,RET or ROS1 and are currently the most promising approach for treating NSCLC patients.The Cancer Genome Atlas Research Network found that EGFR mutations are more frequent among female lung adenocarcinoma patients,whereas mutations in RNA binding motif protein 10(RBM10)are more common among male patients.RBM10 is a member of the RNA-binding protein(RBP)family,is located at position p11.23 of the X-chromosome,and is known for its role in mRNA splicing.Initial functional studies have demonstrated that RBM10 can promote apoptosis and suppress cell proliferation.Furthermore,its expression was found to be correlated with increased expression of the proapoptotic protein BAX and the tumor suppressor protein TP53 in breast cancer.These findings suggest RBM10 as a potential tumor suppressor.However,insufficient research has focused on RBM10-related signaling pathways,which would explain the mechanism of its tumor-suppressing effect.To elucidate the underlying molecular mechanism,we previously performed an Affymetrix GeneChip Primeview Human cDNA microarray analysis.A total of 690 genes that were regulated by RBM10 expression in A549 cells were identified,of which 304 were upregulated and 386 were downregulated.Among these,RAP1 A expression was the most downregulated(63.5%);thus,we explored its downstream signaling pathway.RAP1 A is a type of Ras-associated protein 1(RAP1),with 95% sequence homology to RAP1 B.It is a significant regulator and mediator of Ras functions,and its activation has been linked to a variety of cancers.EPAC is a guanine nucleotide exchange factor(GEF)for the RAP1,which activates RAP1 by GDP-GTP exchange.Agonists and antagonists selective for EPAC have been developed and can be used for further studies on the activation of RAP1,which will increase our understanding on the signaling pathway.In this study,we aimed to explore the effect of RBM10 overexpression and knockdown on the proliferation of lung adenocarcinoma cells as well as the affected downstream pathways by assessing the expression of several regulatory proteins that have previously been identified as being modulated by RBM10.We then sought to verify the effect of RBM10 overexpression and inhibition in vivo in BALB/c mice.Methods:1.A549,H1299 and BEAS-2B cell proteins were extracted,and the expression of RBM10 in non-small cell lung cancer cells and bronchial epithelial cell line BEAS-2B cells was detected by Western Blot.2,A549 and H1299 cells were stably transfected with lentivirus and silenced RBM10 gene.Cell proliferation was detected by CCK8 method and cell clone formation assay.Apoptosis level and cell cycle were detected by flow cytometry.3.RAP1-GTP pull-down method was used to detect the effect of overexpression and silencing RBM10 on RAP1 activation,and by administering RAP1 upstream EPAC-specific activator 8-pCPT-2’-O-Me-cAMP and inhibitor ESI-09 changes in RAP1 activation and cell proliferation were observed.The expressions of key MAPK/ERK,P38 MAPK and AKT pathway proteins and their phosphorylated proteins in the downstream of RAP1 were detected by Western Blot,and the Changes in protein expression was observed by administering 8-pCPT-2’-O-Me-cAMP and ESI-09.4.Overexpressing and silencing RBM10 gene of A549 cells were subcutaneously injected into the axillary fossa of nude mice by lentiviral stable transfection technique.The long diameter and short diameter of the transplanted tumor were measured with vernier calipers every three days,and the volume of the transplanted tumor was calculated.Tumor growth curve was drawn.Nude mice were sacrificed under anesthesia 27 days after tumor implantation.The transplanted tumor tissues were removed,and the long diameter and short diameter were measured to calculate the volume of the transplanted tumor.The expression of AKT and CREB proteins and their phosphorylation were detected by Western Blot.Result:1.With BEAS-2B as control,the relative expression of RBM10 protein in non-small cell lung cancer cell lines(A549,H1299)was significantly decreased(P<0.05).2.In the non-small cell lung cancer cell lines A549 and H1299,overexpression of RBM10 significantly slowed cell proliferation,and silencing RBM10 significantly promoted cell proliferation(P<0.05).The number of clones overexpressing RBM10 was significantly decreased,and the number of clones of silencing RBM10 was significantly increased(P<0.05).There was no difference in the number of early and late apoptotic cells overexpressing or silencing the RBM10.After silencing RBM10 in A549 cells,G0/G1 phase cells were significantly decreased,while H1299 cells were significantly reduced in S phase cells after silencing RBM10,and G2/M phase was significantly increased(P<0.05).It was further confirmed that RBM10 can arrest the A549 cell cycle in the G0/G1 phase and arrest the H1299 cell cycle in the S phase.3.The expression of GTP-RAP1 protein overexpressing RBM10 was significantly decreased in A549 cells.The expression of GTP-RAP1 protein was significantly increased after stimulation with EPAC-specific agonist 8-pCPT-2’-O-Me-cAMP.Proliferation was significantly accelerated(P<0.05).The expression of GTP-RAP1 protein in silencing RBM10 was significantly increased.The protein expression of GTP-RAP1 was significantly decreased and the cell proliferation was significantly slowed down by EPAC-specific inhibitor ESI-09(P<0.05).Overexpression and silencing of the RBM10 had no effect on phosphorylation of key proteins ERK1/2 and P38 in the downstream MAPK/ERK and P38 MAPK pathways.Overexpression of RBM10 significantly reduced the phosphorylation of downstream AKT and CREB,and the phosphorylation of AKT and CREB was significantly increased after stimulation with 8-pCPT-2’-O-Me-cAMP(P<0.05).Silencing of RBM10 significantly increased the phosphorylation of downstream AKT and CREB,and the phosphorylation of AKT and CREB was significantly decreased after stimulation with ESI-09(P<0.05).4.The transplanted tumor model was successfully established by subcutaneous injection of A549 cells overexpressing and silencing RBM10 in nude mice.Compared with the negative control group,overexpression of RBM10 significantly slowed the growth of transplanted tumors,and silencing RBM10 significantly accelerated the growth of transplanted tumors(P<0.05).Compared with the negative control group,the volume of the transplanted tumor overexpressing the RBM10 was the smallest,while the volume of the transplanted tumor with the silent RBM10 was the largest(P<0.05).Overexpression of RBM10 xenografts significantly reduced the phosphorylation of downstream AKT and CREB,and silencing RBM10 xenografts significantly increased the phosphorylation of downstream AKT and CREB.Conclusion:1.Compared with bronchial epithelial cells BEAS-2B,the expression of RBM10 in non-small cell lung cancer cell lines A549 and H1299 was significantly decreased,suggesting that RBM10 is associated with non-small cell lung cancer.2.Overexpression and silencing of RBM10 demonstrated that RBM10 inhibits the proliferation of A549 and H1299 cells,and RBM10 does not inhibit cell proliferation by blocking cell cycle,suggesting that RBM10 is involved in non-small cell lung cancer cells may play a role in tumor suppression.3.Overexpressing and silencing RBM10 in A549 cells confirmed that RBM10 inhibited the activation of downstream RAP1 protein,and the upstream RAP1-specific agonists and inhibitors of RAP1 restored the changes of downstream AKT and CREB protein phosphorylation,thus indicating that RBM10 passes downstream RAP1,AKT.And CREB signaling pathways inhibit cell proliferation.4.RBM10 inhibited the growth of xenografts in nude mice by inhibiting the growth of nude mice by subcutaneous injection of overexpression and silencing RBM10 in A549 cells,and inhibited the phosphorylation of downstream AKT and CREB.These results provide a solid theoretical basis for understanding the mechanism of action of RBM10 in tumorigenesis and development,and also provide new molecular targeted therapy for tumors.