Research On The Expression And Function Of EphA2 In Lung Adenocarcinoma And Its Relationship With Prognosis

BackgroundLung adenocarcinoma is one of the malignant tumors with the highest morbidity and mortality in China.Although many patients have achieved temporary satisfactory curative effects after surgery,postoperative recurrence has always been the most important factor in the death of lung adenocarcinoma.There is a need for a biomarker that can play an important role in predicting postoperative recurrence of lung adenocarcinoma.EphA2 plays an important role in the progression of multiple malignancies but is understudied in lung adenocarcinoma.Objective:To study the expression of EphA2 in lung adenocarcinoma tissues and cells and its relationship with clinical characteristics and prognosis,and to establish a prediction model of postoperative recurrence of lung adenocarcinoma based on the expression level of EphA2.To investigate the effect of EphA2 gene on the proliferation,migration and invasion of lung adenocarcinoma cells,as well as the effect on epithelialmesenchymal transition of lung adenocarcinoma and xenografts in mice.Methods:The surgically resected paraffin specimens(cancer tissue and paracancerous tissue)of 135 patients with lung adenocarcinoma who were operated in our hospital from July1,2016 to October 1,2016 were collected.The expression levels of EphA2 in cancer and adjacent tissues were detected and analyzed by WB and immunohistochemistry.Chi-square test analysis investigated the relationship between the expression of EphA2 and the clinical characteristics of each patient.With RFS as the end point of the study,the adverse factors affecting postoperative recurrence were analyzed by COX regression and Kaplan-Meier survival analysis.Based on the results of multivariate COX regression analysis,a nomogram was constructed to establish a predictive model for predicting postoperative recurrence of patients,and the efficacy of the predictive model was tested with a calibration curve.Lentiviral infection of lung adenocarcinoma cells was used to obtain EphA2 knockdown A549 cells.The knockdown efficiency was tested by q RT-PCR and the expression level of EphA2 after knockdown was detected by WB.The effect of HCS proliferation assay on cell proliferation;the effect of cell invasion assay and scratch assay on the migration and invasion of lung adenocarcinoma cells;the effect of flow cytometry on cell cycle;WB detection of EMT-related marker expression;To establish a mouse model of lung adenocarcinoma to detect the effect of EphA2 on tumor size in mice.Results:1.WB and immunohistochemical results indicated that the expression of EphA2 in lung adenocarcinoma tissues was higher than that in adjacent normal tissues(P<0.05).2.Correlation analysis showed that the expression of EphA2 in lung adenocarcinoma was associated with visceral pleural invasion(P=0.012),TNM stage(P=0.011)and recurrence(P=0.001).However,there was no significant correlation with age(P=0.893),gender(P=0.789),smoking(P=0.068),tumor diameter(P=0.401),degree of differentiation(P=0.183)and lymph node metastasis(P=0.33).3.In the univariate COX regression analysis,the high expression of EphA2 in LUAD tissue was a risk factor for the poor prognosis of patients(HR=3.04,95% CI=1.818-5.038,P<0.001).In addition,tumor diameter was large(HR=1.974,95%CI=1.171-3.237,P=0.01);poor differentiation(HR=2.313,95%CI=1.389-3.852,P=0.001);visceral pleural invasion Presence(HR=2.701,95% CI=1.538-4.754,P=0.001);advanced TNM stage(HR=3.402,95% CI=2.041-5.669,P<0.001)both indicated poor prognosis.No statistically significant associations were found for gender,smoking status,and presence or absence of lymph node metastases.4.In multivariate COX regression analysis,tumor diameter(HR=2.132,95%CI=1.204-3.775,P=0.009);differentiation degree(HR=1.801,95% CI=1.033-3.319,P=0.038);TNM stage(HR=2.717,95% CI=1.552-4.757,P<0.001)and high expression of EphA2 in cancer tissues(HR=3.13,95% CI=1.813-5.404,P<0.001).These were independent factors to affect the prognosis of these 135 patients with lung adenocarcinoma.The Kaplan-Meier survival curve indicated that the recurrence-free survival rate of patients with high expression of EphA2 was lower than that of patients with low expression(P<0.0001).5.The nomogram based on the results of multivariate Cox analysis was used to establish a model for predicting postoperative recurrence,and the calibration chart was used to test and showed good predictive performance.The test efficacy index c-index=0.755.6.WB results suggested that the expression of EphA2 in A549 cells was higher than that in PC9 and H1299.q RT-PCR detection showed that the expression of EphA2 m RNA in A549 cells knocked down EphA2 was lower than that in the control group(P<0.01).7.The results of the HCS cell proliferation experiment indicated that the proliferation rate of A549 cells was lower than that of the control group after knockdown of EphA2(P<0.01).8.The results of cell scratch experiments suggest that the migration ability of A549 cells after knockdown of EphA2 is lower than that of the control group(P<0.001).9.The results of the Transwell experiment showed that the invasive ability of A549 cells was decreased compared with the control group after knockdown of EphA2(P<0.001).10.After knocking down EphA2,the expression of E-cadherin factor was enhanced,while the expression of vimentin and Snail was inhibited(P<0.01),indicating that EphA2 may promote EMT in lung adenocarcinoma.11.The results of mouse xenograft experiments indicated that the volume of xenografted tumors in mice injected with EphA2 knockdown A549 cells was significantly smaller than that in control mice(P<0.05).Conclusions:The expression of EphA2 in lung adenocarcinoma tissue is higher than that in adjacent normal tissue.The high expression of EphA2 in lung adenocarcinoma tissue is correlated with clinical features VPI and TNM staging and indicates poor prognosis.A predictive model for postoperative recurrence of lung adenocarcinoma was established It has high test power.After knockdown of EphA2 gene,the proliferation,migration and invasion ability of lung adenocarcinoma cells were inhibited.EphA2 may be involved in promoting the recurrence of lung adenocarcinoma and promoting the growth of xenografts in mice through EMT.