The Role And Mechanism Of Rap2b Promotes Proliferation,Migration,and Invasion Of Lung Cancer Cells

Objective:Non-small cell lung cancer(NSCLC)has been regarded as the leading cause of malignant tumors related mortality.The treatment of NSCLC has made great progress in recent years,especially for gene targeting therapy,but the 5 year survival rate of NSCLC is only 15%.Therefore,it is of great significance to explore the pathogenesis of NSCLC and to find new molecular targets.The expression of Rap2b is upregulated in many types of tumors,but the expression of Rap2b in NSCLC and its potential mechanism are still unclear.The purpose of this study is to explore the correlation between Rap2b and clinicopathological parameters and prognosis in NSCLC,and to investigate the effect of Rap2b on the phenotype of NSCLC cells and the related mechanism.Methods:(1)90 cases of non-small cell lung cancer and adjacent tissues were collected.The expression of Rap2b was detected by immunohistochemistry staining(IHC),and the correlation with the clinicopathological parameters and prognosis was analyzed.(2)Western blotting method was used to detect the expression level of Rap2b in lung cancer tissues and corresponding adjacent tissues.Western blotting method was used to detect the expression of Rap2b in lung cancer cell lines H460,H1299,A549 and human fetal lung cell lines MRC-5.(3)RNA interference technique was used to silence Rap2b,and the expression of Rap2b was detected by RT-PCR and Western blot respectively.(4)The effects of Rap2b gene knockdown on proliferation,colony formation,migration,invasion and cell cycle of lung cancer cells were detected by CCK-8 test,colony formation test,Wound healing assays,Transwell experiment and flow cytometry detection.(5)Western blot was used to detect the expression of p-AKT,p-ERK,of proliferation related proteins.The changes of metastasis associated protein Snail,MMP-2 and MMP-9 were detected by Western blot.Results:(1)Immunohistochemical staining was performed to determine the expression of Rap2b in 90 NSCLC paraffin-embedded slices.We found that Rap2b was seen predominantly in the cell membrane and plasma membrane.Moreover,we analyzed the association between Rap2b expression and clinicopathologic parameters of the patients,which were summarized.High Rap2b expression shows a significant correlation with TNM stage(p=0.017<0.05),tumor diameter(p=0.015<0.001),and lymph node metastasis(P=0.018<0.001),which was no statistical relationship between the presence of Rap2b with gender,age,pathological type and tumor differentiation.(2)Rap2b was significantly higher in tumor tissues than the adjacent non-cancerous lung tissues by Western blotting.The expression of Rap2b protein in lung cancer cell lines H460,H1299,and A549 was higher than that in human fetal lung cells by Western blotting.(3)The results of RT-PCR and Western blot assay showed that the expression of Rap2b mRNA and protein was downregulated in A549 and H1299 cells after Rap2b silencing.(4)The CCK-8 experiments showed that Rap2b gene silencing of A549 and H1299 cell proliferation was inhibited;the cloning formation experiments showed that Rap2b was able to inhibit the cloning of A549 and H1299 cells after the reduction of Rap2b.The wound healing assay and Transwell assay showed that Rap2b gene could inhibit the invasion and migration ability of A549 and H1299 cell significantly after the reduction of Rap2b.The results of flow cytometry showed that interference with the expression of Rap2b had no effect on the cell cycle.(5)Western blotting assay showed that the expression of p-AKT and p-ERK was down regulated,and the expression of Snail,MMP-2 and MMP-9 was also down regulated.Conclusions:(1)Rap2b was up-regulated in NSCLC tissue and the expression of Rap2b was positively correlated with TNM stage,tumor diameter and lymph node metastasis.High expression of Rap2b predicted poor prognosis of NSCLC patients.Rap2b is a independent predictor of NSCLC.(2)After the interference of Rap2b gene,the proliferation ability of lung cancer cells can be inhibited by AKT and ERK signaling pathway.After Rap2b gene interference,the metastasis ability of lung cancer cells can be inhibited by Snail and MMP family.Targeting Rap2b and the signal pathway of Rap2b might become a potential treatment of NSCLC.