The Molecular Mechanism Study On The RBM5 Gene Reversing Acquired Resistance To EGFR-TKI In Lung Adenocarcinoma

Background: The latest surveys show that morbidity and mortality of lung cancer are ranked highest among malignant tumors in China,which threatens to human health and brings about heavy burden for society and families.In the various classifications of lung cancer,the incidence of NSCLC accounts for about 70%-80%,and about 80% of those patients is already in the middle or late stage when they are newly diagnosed.Therefore,the molecular mechanism of lung cancer has become one of the hotspots of researches,in which EGFR is one of those targets.EGFR-TKI is the small molecular targeted drugs,represented by Gefitinib,characterized by its specific,high efficiency and few side effects,which has been widely popularized and used in clinical practice.The results of several large-scale III phase clinical trials have proved that the Objective Response Rate of NSCLC patients can reach 58% to 83% and the Progression Free Survival can be extended to 10-16 months after using EGFR-TKI.However,acquired resistance to EGFR-TKI will occur and result in rapid progress and deterioration after a period of medication.The acquired resistance limits the use of the small molecular targeted drug.Therefore,researching on the molecular mechanism of acquired resistance to EGFR-TKI has become a hotspot of targeted therapy for lung cancer.As a tumor suppressor,RBM5 is mainly distributed in the nucleus.It can regulate the alternative splicing of related genes by recognizing and binding m RNA,and thus participates in the regulation of cell cycle and apoptosis.Moreover,RBM5 has a close relation with lung cancer.Our team has already proved that the overexpression of RBM5 could reverse the drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin,and hinder the Wnt/β-cateninsignaling pathway.Therefore,we speculate that RBM5 may be associated with acquired drug resistance to EGFR-TKI,and expression decrease of RBM5 may be one of the mechanisms.Moreover,regulation expression of RBM5 may be a new target for lung cancer therapy.To verify this hypothesis,we use human lung adenocarcinoma cell line PC9 and Gefitinib resistant cells PC9/AB2 as the object,and use of RBM5 and Wnt/β-cantenin pathway as targets,to observe the effect of RBM5 on acquired resistance to EGFR-TKI by enhancing the expression of RBM5 gene and combining application of Wnt/β-catenin pathway specific inhibitor ICG-001 in vivo and in vitro experiment.Our aim is to explore the relationship between the RBM5,Wnt/β-catenin pathway and the EGFR pathway,as well as the possible mechanism of the occurrence and reversal of acquired resistance to EGFR-TKI.Method: 1.Difference between drug resistant cell PC9/AB2 and its parent cell PC9.(1)CCK8 method was used to compare the sensitivity of human lung adenocarcinoma PC9 cells and the drug resistant cell PC9/AB2 to Gefitinib.(2)The morphological differences of PC9 cells and PC9/AB2 cells were observed by inverted microscope.(3)RT-PCR was used to detect the level of RBM5 gene expression in PC9 cells and PC9/AB2 cells.(4)Western blot method was used to detect the expression level of RBM5 protein in PC9 cells and PC9/AB2 cells.(5)Western blot methods was used to detect the expression level of Wnt/β-catenin pathway and EGFR pathway related protein in PC9 and PC9/AB2 cells.2.The mechanism of overexpression of RBM5 on acquired resistance to EGFR-TKI in PC9/AB2 cells.(1)pc DNA3.1-RBM5 was identified by agarose gel electrophoresis.(2)Identification of the efficiency of lentivirus infection.A.The expression of green fluorescent protein was observed under the fluorescence microscope to judge the efficiency of infection.B.RT-PCR was used to detect the infection efficiency of lentivirus vectors with RBM5 gene target fragments.C.Western blot methods was used to detect the infection efficiency of lentivirus vectors with RBM5 gene target fragments.(3)The CCK8 assay was used to detect the sensitivity to Gefitinib of PC9/AB2 cells expressing RBM5.(4)Transwell methods was used to detect the cell invasiveness in each treatment group.(5)Cell wound test to evaluate the cell migration ability of each treatment group.(6)Flow cytometry method was used to detect the cell cycle and apoptosis in each treatment group after overexpression of RBM5.(7)RT-PCR method was used to detect the expression level of related genes in the Wnt/β-catenin pathway and EGFR pathway.(8)Western blot method was used to detect the expression level of Wnt/β-catenin pathway and the related protein of EGFR pathway.3.Effect and mechanism of overexpression of RBM5 on the growth of PC9/AB2 cell xenografts in BALB/c nude mice(1)The weight of the nude mice was weighed and the volume of the transplanted tumor was measured and the growth curve of the tumor was drawn.(2)RT-PCR method was used to detect the expression of RBM5,β-catenin and EGFR gene in xenografts in nude mice.(3)Immunohistochemical staining of subcutaneous tissue in nude mice to evaluate the expression of RBM5、β-catenin and EGFR protein.Result: 1.The sensitivity of PC9/AB2 cell to Gefitinib was significantly lower than PC9 cells that is sensitive to Gefitinib(P<0.001).At the same time,we observed that the size of PC9/AB2 cells became larger and the morphology was irregular under the inverted microscope.RT-PCR method and Western blot method showed that compared with PC9 cells,the expression of RBM5 gene and protein decreased in PC9/AB2 cells(P<0.01),and the expression of Wnt/β-catenin pathway and EGFR pathway related protein increased(P<0.05).2.To verify the plasmids with RBM5 gene,we use agarose gel electrophoresis,the inverted microscope,RT-PCR method and Western blot method.Finally,our conclusion is that the virus vector with RBM5 gene has infected PC9/AB2 cells,andRBM5 gene has expressed steadily(P<0.001).We establish the cell line with highly expression of RBM5 in vitro.3.Overexpression of RBM5 can increase the sensitivity of PC9/AB2 cells to Gefitinib(P<0.001),and inhibit cell invasion and migration,and induce G1 phase arrest and promote cell apoptosis(P<0.05).Wnt/β-catenin pathway specific blocker ICG-001 can also increase the sensitivity of PC9/AB2 cells to Gefitinib,and reduce cell invasion and migration ability,and promote cell apoptosis.The combined application of ICG-001 can enhance the biological effects of overexpression of RBM5.4.Overexpression of RBM5 can downregulate the expression of Wnt/β-catenin pathway related factors,for example c-Jun and c-Myc.Moreover,it also can upregulate the expression of GSK-3β,and downregulate the phosphorylation level of EGFR and AKT(P<0.05).The specific blocker of Wnt/β-catenin pathway can reduce the expression level of EGFR related factors(EGFR,Akt,Erk)(P<0.05).5.The subcutaneous tumor model of nude mice was successfully established by the cell overexpressed RBM5,and RBM5 could be expressed steadily in the transplanted tumor(P<0.01).Overexpression of RBM5 could inhibit the growth of tumor transplanted subcutaneously in nude mice with,and downregulate the Wnt/β-catenin pathway and the EGFR pathway(P<0.05).At the same time,when the Wnt/β-catenin pathway was downregulated,the expression level of EGFR also decreased(P<0.05).In addition,the Wnt/β-catenin pathway specific blocker,ICG-001,can increase the sensitivity of the drug resistant cells to Gefitinib and inhibit the growth of the transplanted tumor.Combined use of ICG-001 can enhance the tumor suppressive effect of RBM5 overexpression.Conclusion: 1.RBM5 is associated with acquired resistance to EGFR-TKI in human lung adenocarcinoma,and expression decrease of RBM5 may be one of the acquired drug resistance mechanisms.2.RBM5 may be a key regulator of Wnt/β-catenin and EGFR cross pathway.It may delay or reverse acquired resistance to EGFR-TKI by blocking the conduction of EGFR signaling pathway by accommodating the Wnt/β-catenin pathway.3.Wnt/β-catenin pathway is associated with EGFR-TKI acquired resistance.Down regulating Wnt/β-catenin pathway can inhibit EGFR pathway and increase thesensitivity of drug-resistant cells to EGFR-TKI.Combination application Overexpression of RBM5 and ICG-001 have a synergistic effect of reversing of acquired resistance to EGFR-TKI.Therefore,Wnt/β-catenin pathway blocker ICG-001 combined with RBM5 overexpression may be an effective way to treat lung adenocarcinoma and reverse acquired resistance to EGFR-TKI.In conclusion,our research has demonstrated that RBM5 is closely related to acquired resistance to EGFR-TKI,and it may be one of the important regulatory factors.The regulation of the expression of RBM5 may be a new target for the treatment and reversing of drug resistance in lung cancer,however,further studies on the molecular mechanisms are needed.It also provides a new theoretical basis and perspective for reversing acquired resistance to EGFR by combination of overexpression of RBM5 and the inhibitor of Wnt/β-catenin pathway.