Studies On The Expression Of RBP4 And Its Possible Mechanisms In Non-small Cell Lung Cancer

Objective:We discussed the expression of RBP4 and its malignant biological behavior and its possible molecular mechanism in non-small cell lung cancer.Methods1.Investigated the expression of RBP4 in patients with non-small cell lung cancer.(1)Bioinformatics analysis:Using the GEPIA and UALCAN databases,analyze the expression of RBP4 in non-small cell lung cancer.(2)By collecting fresh specimens of 56 patients with NSCLC(29 adenocarcinomas and 27 squamous cell carcinomas),we compared the expression of RBP4 in cancerous and normal tissues by quantitative real-time PCR and collected clinical data to analyze the correlation between RBP4 and clinicopathological characteristics of patients with NSCLC.2.Investigated the biological behaviors of non-small cell lung cancer cells such as proliferation,migration,invasion,apoptosis,and cell cycle after overexpression of RBP4,and analyzed their molecular mechanism.(1)Using quantitative real-time PCR detected the expression of RBP4 between different NSCLC cell lines and normal cell lines,and choosing the tumor cell line with the largest difference in expression compared to normal cell lines as the experimental group;(2)Transfecting lung cancer cells used RBP4 overexpression plasmid,and verifing the transfection efficiency of the cell lines with an inverted microscope and qRT-PCR;(3)Using the CCK-8 assays to detect the proliferation ability of NSCLC cells after over-expression of RBP4;(4)Transwell experiment was used to observe the migration and invasion ability of NSCLC cells after over-expression of RBP4;(5)Flow cytometry was conducted to detect the apoptosis rate and cell cycle of NSCLC cells after overexpression of RBP4;(6)Western blot was used to detect the protein expression levels of Bcl-2,Bax,CD44 and other genes after over-expression of RBP4.3.Statistical analysisIn this study,SPSS 24.0 software was used for statistical analysis.Chi-square test was used to analyze the relationship between RBP4 expression and demographic and clinicopathological features of NSCLC patients.The results of qRT-PCR and functional experiments were performed using two independent sample t tests(Independent-Samples T Test),P<0.05 was considered statistically significant.Results:1.RBP4 expression was significantly lower in NSCLC patientsBioinformatics analysis found that compared with normal tissues,RBP4 was under-expressed in lung adenocarcinoma and lung squamous cell carcinoma,with fold differences of 1.9 and 2.7 times,respectively.There was no significant correlation between abnormally expressed RBP4 and survival prognosis in patients.The qRT-PCR results showed that in 56 NSCLC patients,the expression of RBP4 mRNA in cancerous was significantly lower than that in normal tissues,and the difference was statistically significant(P<0.05),which was consistent with the results of the database.However,the clinicopathological characteristics of patients were not significantly correlated with the gene.2.Relationship between RBP4 and biological function of non-small cell lung cancer A549 and SK-MES-1 cells(1)The results of qRT-PCR showed that RBP4 was abnormally expressed in different lung cancer cell lines compared with BEAS-2B,and its expression was significantly lower in A549 and sk-mes-1 cells.Therefore,A549 and SK-MES-1 were used as the experimental group cells of this project.(2)Transfection of lung cancer cells with over-expression of RBO4,qR-TPCR results showed that the expression of RBP4 mRNA in the experimental group was significantly higher than that of the corresponding control group,the difference was statistically significant(P<0.05),indicating that the gene was successfully overexpressed.(3)CCK8 results showed that compared with the control group,the proliferation ability of A549 and SK-MES-1 cells was significantly reduced after RBP4 overexpression(P<0.05),indicating that RBP4 can inhibit the proliferation of cells.(4)Transwell results showed that compared with the control group,with or without matrigel,the transmembrane number of A549 and SK-MES-1 cells were significantly reduced after RBP4 overexpression(P<0.05),indicated that RBP4 could inhibit migration and invasion ability of lung cells.(5)The results of flow cytometry showed that compared with the control group,the apoptosis rate of A549 and SK-MES-1 cells with the over-expressed RBP4 increased(P<0.05),indicating that RBP4 can promote lung cancer cell apoptosis.(6)Flow cytometry results showed that compared with the control group,the cell cycle A549 with overexpressed RBP4 was arrested in the G0/G1 phase,while the SK-MES-1 cell cycle was arrested in the M phase,the differences were statistically significant(P<0.05),indicating that RBP4 can inhibit cell proliferation by blocking the cell cycle.(7)Western blot results showed that compared with the control group,Bcl-2 and CD44 protein expression in lung cancer cells was significantly down-regulated and Bax expression was up-regulated after over-expression of RBP4.Conclusions:1.The RBP4 expression was low in NSCLC,but the abnormal expression of RBP4 had no significant assessment and prognosis-estimation for NSCLC patients.2.RBP4 played an inhibitory role in lung adenocarcinoma A549 and lung squamous cell carcinoma SK-MES-1 cells,and provided a theoretical basis for further studying the role of RBP4 in the development of non-small cell lung cancer.3.RBP4 may be involved in the development of NSCLC by inhibiting the anti-apoptotic protein Bcl-2,promoting the expression of the apoptotic protein Bax,and down-regulating the expression of tumor stem cell CD44 protein.