Effects Of RBM4 On Malignant Biological Behavior Of Gastric Cancer And Its Molecular Mechanism

Background:Gastric cancer remains as the second leading cause of cancer death worldwide,and advanced gastric cancer has a high rate of metastasis and recurrence.Traditional surgery,radiotherapy and chemotherapy are poor efficacy,molecular targeted therapy has not achieved significant treatment effect.Therefore,it is important to find a new target for gastric cancer and to effectively intervene,which is of great significance for clinical treatment,prognosis and improvement of patient survival.RNA-binding motif 4(RBM4)is a splicing factor with multiple functions,including regulation of alternative splicing,translation control and RNA silencing.Early studies suggest that RBM4 is up-regulated in leukemic cells.In recent years,it has been reported that RBM4 expression is down-regulated in a variety of solid tumors,suggesting that RBM4 gene is closely related to the development of tumor,but the specific mechanism and molecular mechanism is still unclear.Objective:First,to observe the RBM4 expression in gastric cancer and gastric benign lesions,and analyze the relationship of RBM4 expression and clinicopathological factors and prognosis.Second,to observe the influence of RBM4 on malignant biological behavior of gastric cancer cell proliferation,apoptosis,migration and invasion in vivo and in vitro.Then,to investigative the molecular mechanisms of RBM4 on malignant biological behaviors of gastric cancer.Finally clarify RBM4 can be used as a new molecular target in gastric cancer for molecular targeted therapy.Methods:1.to observe the RBM4 expression in gastric cancer and gastric benign lesions,and analyze the relationship of RBM4 expression and clinicopathological factors and prognosis.The experimental methods are as follows:(1)The protein expression of RBM4 in tissue microarray(TMA)was detected by immunohistochemistry.(2)The mRNA expression of RBM4 was detected by qRT-PCR in 25 cases of postoperative gastric cancer tissues and adjacent tissues.2.to observe the influence of RBM4 on malignant biological behavior for gastric cancer cell proliferation,apoptosis,migration and invasion in vivo and in vitro.We Used RBM4 overexpressing lentiviral vector and targeting silencing RBM4 vector and then perform the following experiments:(1)The expression of RBM4 in gastric cancer cells was observed by Western Blot and flow cytometry,and then the low expression and high expression cell lines of RBM4 in gastric cancer were screened out.(2)The effect of RBM4 on the proliferation of gastric cancer cells was observed by CCK-8.(3)To observe the effect of RBM4 on the apoptosis of gastric cancer cells.(4)To observe the effect of RBM4 on the migration of gastric cancer cells by scratching experiment and transwell chamber migration experiment.(5)To investigate the effect of RBM4 on gastric cancer cells invasion by transwell chamber invasion experiment.(6)To observe the effect of RBM4 on subcutaneous xenograft growth of gastric cancer cell.3.to investigative the molecular mechanisms of RBM4 in malignant biological behaviors of gastric cancer.The experimental methods are as follows:(1)The protein expression of p53 and Bcl-2 were detected by immunohistochemistry in subcutaneous xenografts of gastric cancer.(2)Effects of RBM4 on MAPK(mitogen activated protein kinase)Pathway protein expression in gastric cancer cells by western blot.Results:1.The positive expression of RBM4 protein in gastric cancer tissues was 43.8%,while the positive expression of RBM4 protein in gastric benign lesions such as chronic gastritis,intestinal metaplasia,low grade neoplasia,high grade neoplasia and adjacent tissues were 58.6%and 56.0%,54.2%,51.9%and 63.1%respectively.Compared with the adjacent tissues,the mRNA expression of RBM4 in gastric carcinoma was down-regulated by 64.3%.There was a significant correlation between the expression of RBM4 protein and the differentiation of gastric carcinoma(P<0.001),lymph node metastasis(P=0.026),TNM status(P=0.014)and distant metastasis(P=0.036).Patients with low or no expression of RBM4(P<0.001,CI=0.315-0.710)and TNM III and IV(P<0.001,CI=4.757-11.166)had a worse overall survival(OS)and none disease-free survival(DFS).multivariate analysis showed that RBM4 was an independent prognostic factor.2.The expression of RBM4 protein in normal gastric mucosal epithelial cell was 52.9%,and the expression levels of RBM4 protein in gastric cancer cells MKN28,HGC27,MKN45,BGC823 and MGC803 were 43.52%,25.37%,20.36%,17.34%and 45.95%respectively.BGC823 was screened as gastric cancer cell line of RBM4 low expression,MGC803 was screened as gastric cancer cell line of RBM4 high expression.RBM4 overexpression inhibited the proliferation of BGC823 cells,RBM4 interference promoted MGC803 cell proliferation ability.The apoptotic capacity of BGC823 cells was increased by 431%after RBM4 overexpression and the apoptotic capacity of MGC803 cells was down-regulated by 44.5%after RBM4 interference.In the scratch experiment,the migration ability of BGC823 cells was decreased by 71.2%after RBM4 overexpression,and the migration ability of MGC803 cells was increased by 114.3%after RBM4 interference.In the transwell chamber migration experiment,the migration ability of BGC823 cells was decreased by 55.6%after RBM4 overexpression,and the migration ability of MGC803 cells was increased by 95.1%after RBM4 interference.In the transwell chamber invasion experiment,the invasive ability of BGC823 cells was down-regulated by 51.2%after RBM4 overexpression,and the invasive ability of MGC803 cells was increased by 77.1%after RBM4 interference.Overexpression of RBM4 can significantly inhibit the growth of BGC823 gastric cancer cells in nude mice.3.The expression level of p53 protein in the subcutaneous xenograft tumor of gastric cancer was increased by 680%and the expression level of Bcl-2 protein was down-regulated by 86.7%after RBM4 overexpression.The expression levels of p-ERK1/2,p-JNK and p-p38 protein in BGC823 cells after RBM4 overexpression were down-regulated by 66%,20%and 48%,respectively.While the expression levels of p-ERK1/2,p-JNK and p-p38 protein in MGC803 cells after RBM4 interference were up-regulated by 225%,55%and 193%,respectively.Conclusion:The expression level of RBM4 in gastric carcinoma was down-regulated,and the down-regulation of RBM4 expression was closely related to gastric cancer tissue differentiation,lymph node metastasis,TNM status,distant metastasis and poor prognosis.RBM4 could inhibit the proliferation,migration and invasion of gastric cancer cells,and promote the apoptosis of gastric cancer cells and inhibit the growth of subcutaneous xenografts of gastric cancer.RBM4 may affect the malignant biological behavior of gastric cancer cells through p53/Bcl-2 and MAPK-dependent pathways.The results of this study suggest that RBM4 is expected to be a new target for clinical targeted treatment of gastric cancer.