Study On TPX2 And MMP12 In Molecular Classification And Progression For Non-small Cell Lung Cancer

Part 1.Study on the association between a predictive model based on TPX2 and MMP12 and prognosis for non-small cell lung cancerObjective 1.To explore the differential expression profile of non-small cell lung cancer(NSCLC)in public Datasets.Then analysis the differential expression genes in NSCLC tissues,construct a survival prediction model for prognostication in patients with NSCLC.Methods 1.Searching and filtering expression microarray data in public datasets,obtaining the suitable microarray data related to NSCLC.2.The gene expression profile of NSCLC was obtained by comparing those microarray data and comprehending their design of experiment.2.QRT-PCR and IHC method was used to verify those genes expression in NSCLC.3.Cox regression analysis was performed to determine significant prognostic factors.4.The survival prediction model was constructed by logistic regression analyses in 152 NSCLC cases.6.The predictive performance of the model was assessed by Kaplan-Meier survival analysis.7.Receiver operating characteristic(ROC)curve was used to compare the sensitivity and specificity of the prognostic parameters.Results(1)A total of 2 sets of data in GEO database met the experiment required:GSE31552 and GSE18842.(2)A total of 334 and 1856 genes which showed differentially expression levels in GSE31552 and GSE18842 dataset were detected respectively.Among these genes,143 up-regulated genes and 126 down-regulated genes were found in both datasets.(3)The mRNA expression of TPX2 and MMP12 showed significant difference between tumor and normal lung tissue(P<0.05),However,there was no significant expression difference in other four genes(DSG3,SFTPC,TMEM100 and AGER)(P>0.05).The positive protein expression rates of TPX2 and MMP12 were up to 48.7%(74/152)and 58.6%(89/152),respectively.(4)Multivariate Cox proportional hazards regression analysis revealed that TNM stage,tumor grade,TPX2 expression and MMP12 expression were independent predictors for disease-free survival(P<0.05),TNM stage,tumor grade,TPX2 expression and MMP12 expression were independent prognostic factors for overall survival(P<0.05).(5)The prognostic model for disease-free survival was calculated as:Y=3.234*TNM+2.928*Grade+0.026*TPX2+0.025*MMP12.The predictive model for overall survival was calculated as described in the equation:Y=3.223*TNM+3.114*Grade+0.030*TPX2+0.025*MMP12.(6)The postoperative 5-year disease-free survival rate in high-risk group was significantly lower than that in low-risk group(17.6%vs26.2%,P=0.025).The patients in high-risk group had a worse outcome than those in low-risk group,and the postoperative 5-year overall survival rates were 21.9%and 37.7%,respectively(P=0.021).(7)The area under the ROC curve(AUC)value for the disease-free survival model was higher than that for TNM system(0.771(95%CI,0.689-0.853)vs 0.719(95%CI,0.633-0.804)).The AUC value of the survival model was larger than that of TNM stage(0.761(95%CI,0.678-0.844)vs 0.700(95%CI,0.612-0.787)).Conclusion(1)We have developed a predictive model that is based on two gene markers in conjunction with two clinicopathological variables for NSCLC.(2)The model could accurately predict outcomes for NSCLC patients after surgery.Part 2.The effect of TPX2 expression on non-small cell lung cancer cell proliferation,migration and invasion and its underlying mechanismObjective:To investigate the effects of inhibbited TPX2 expression on tumor proliferation,migration and invasion of Non-small cell lung cancer cell and its underlying mechanism.Methods(1)The expression levels of TPX2 mRNA and protein were detected by real-time quantitative PCR and Western blotting methods in NSCLC cell lines including H157、H1299、H1650 and A549 cells.(2)Real-time quantitative PCR and Western blotting were used to identify the NSCLC A549 and H1299 cells untreated or transfected.(3)The effect of TPX2 downregulation on the proliferation of NSCLC A549 and H1299 cells was evaluated by CCK-8 proliferation experiment.(4)The effect of TPX2 downregulation on the migration and invasion of NSCLC A549 and H1299 cells was assessed by transwell chambers experiment.(5)The changes of PI3K-AKT pathway-realted proteins such as PI3K,AKT and p-AKT,migration-related moleculer MMP9 and MMP12 protein expressions was measured by Western blotting in NSCLC A549 and H1299 cells untreated or transfected.(6)IGF-1 agonist experiments were used to evaluate the effects of TPX2 on PI3K/AKT signaling pathway activity and downstream MMP9 and MMP12 expression.Results(1)NSCLC H157、H1299、H1650and A549cells displayed higher TPX2 mRNA and protein levels than that in human bronchial epithelial cell(HBE),and the differences were statistical significance(P<0.05).(2)The levels of TPX2 mRNA and Protein in TPX2 siRNA group were significantly lower than those in control siRNA group,and the differences were statistical significance(P<0.05).(3)Compared with control siRNA cells,TPX2-depleted grew more slowly,and the differences were statistical significance(P<0.05).(4)Migration and invasion of TPX2-depleted A549 and H1299 cells decreased markedly than control siRNA cells,and the differences were statistical significance(P<0.05).(5)TPX2 depletion markedly decreased the expressions of PI3K,p-AKT,MMP9 and MMP12 proteins,and the differences were statistical significance(P<0.05).(6)The agonist IGF-1 could attenuate the inhibition of PI3K/AKT signaling pathway activity and the expression of downstream MMP9 and MMP12 by TPX2-siRNA in A549 and H1299 cells.Conclusion Our results showed that inhibbited-TPX2 expression can inhibit A549 and H1299 cell proliferation,mirgartion and invasion.TPX2 regulateed PI3K/AKT signaling pathway activity and its downstream MMP9 and MMP12 expression was important mechanism for TPX2 to promote invasion and metastasis of non-small cell lung cancer cells.