Effect And Mechanism Of Down-regulated PRRX1 Promoting Cell Proliferation And Inhibiting Apoptosis In Non-small Cell Lung Cancer

Prat I Expression of PRRX1 in non-small cell lung cnacer and its clinical significance Objective: To investigate the expression level of PRRX1 in non-small cell lung cancer,combined with the patient’s clinical feature and to study its clinical significance.Methods:Immunohistochemical analysis was used to determine PRRX1 protein expression in 52 NSCLC tissue samples and 28 cases of non-tumour adjacent tissues.The relationship between PRRX1 expression and clinicopathological variables was examined by chi-square test.The survival rate was acquired from the Kaplan–Meier survivor curve and Cox regression analysis.Results: 1.The level of PRRX1 expression was steady and moderate in non-tumour adjacent tissues.Strong staining was observed in well-differentiated NSCLC tissue sections,weak or moderate staining was observed in moderately differentiated NSCLC tissue sections,and absent or weak staining was observed in poorly differentiated NSCLC tissue sections.2.Low PRRX1 expression levels were significantly associated with T stage,advanced tumour stage and poor tumour differentiation(P=0.002,P=0.019 and P=0.001,respectively).3.Multivariate analysis revealed that N stage,disease stage,and PRRX1 expression levels were significant independent prognostic factors connected with overall survival(Cox regression analysis,P=0.048,P=0.008 andP=0.035,respectively).4.NSCLC patients with low PRRX1 expression levels showed significantly shorter survival times compared to those with high PRRX1 expression levels(log-rank test,P=0.010).Conclusion: The loss of PRRX1 expression correlated with poor tumour differentiation and advanced histopathological stage and T stage.Additionally,low PRRX1 expression is an independent factor related to overall survival.Part II Loss of PRRX1 induced epithelial-mesenchymal transition and cancer stem cell-like properties in A549 cells Objective: To construct lung cancer cells model with silencing PRRX1 gene,and to reveal the relationship between PRRX1 and EMT、CSCs-like properties.Methods:Silence PRRX1 gene in A549 cells with sh RNA technology constructs PRRX1-sh RNA-A549 model.The PRRX1 repression level was tested by Western blot to validate models.MTT assay and soft agar colorimetric assays were used to observe the lung cancer cells proliferation.Phase contrast microscope showed the changes about cellular morphology.The ability of migration and invasion were evaluated by Transwell assays and wound-healing assays.The expression of E-cadherin、V-cadherin and vimentin were analyzed with Western blot and immunofluorescence analysis.Flow cytometric analysis detected the percentage of lung cancer cells CSCs markers.Results: 1.PRRX1 expression level in A549 cells was high,it was appropriate for gene silencing cell model.2.The PRRX1 expression level was low after transfection.The PRRX1-sh RNA-A549 cell model was construct successfully.3.Silencing PRRX1 promoted the ability of proliferation.4.After silencing PRRX1,A549 cells acquired a mesenchymal phenotype.5.Silencing PRRX1 increased the ability of migration andinvasion.6.Up-regulated of vimentin and N-cadherin protein(mesenchymal cell markers)and down-regulated of E-cadherin protein(epithelial cell markers)were investigated by Western blot and immunofluorescence analysis after silencing PRRX1.7.The A549 cells percentage with CD133,CD44 and ALDH1,as CSCs-like A549 cell markers,were 77.6%、4.1% and 1.5%.Conclusion:.After silencing PRRX1,A549 cells underwent EMT,acquired CSCs-like properties,and promoted the abilities of proliferation and invasion.Part III Loss of PRRX1 inhibited A549 cells apoptosis induced by cisplatin through interdicting caspase-3 pathway Objective: To observe A549 cells apoptosis induced by cisplatin after silencing PRRX1,and to reveal the mechanism about PRRX1 and apoptosis pathway.Methods:MTT assay was used to observe the lung cancer cells proliferation inhibition by cisplatin.Flow cytometric analysis detected cells apoptosis rate、cell cycle distribution.JC-1 detected mitochondrial membrane potential.The expression of caspase-3、caspase-9、cytochrome C and Apaf-1 were analyzed with Western blot.After adding PAC-1,MTT assay and flow cytometric assay indicted cell proliferation and apoptosis.Results: 1.The apoptosis of A549 cells was observed after the treatment of different concentrations of cisplatin.MTT results showed that down-regulated PRRX1 gene could resist the inhibitory effect of cisplatin on cell proliferation.The best medical concentration was 20 μg/ml.2.The results of flow cytometry assay showed that down-regulated PRRX1 gene could reduce the apoptosis 3.The results of flow cytometry assay showed that down-regulated PRRX1 gene couldpromote A549 cells to enter G2 phase.4.Mitochondrial membrane potential detection showed that PRRX1 gene could inhibit the decrease of mitochondrial membrane potential.5.Western blotting results showed that down-regulated PRRX1 gene could reduce the expression levels of Caspase3,caspase9,Apaf-1 and cytochrome C.6.After adding PAC-1 in MTT assay and flow cytometric assay,cell proliferation has been inhibited and cell apoptosis increased.Conclusion:.Loss of PRRX1 in A549 cells stabilized the mitochondrial membrane potential and inhibited cell apoptosis induced by cisplatin through interdicting caspase-3 pathway.