The Interaction Between CXCR4 And EGFR And Downstream PI3K/AKT Pathway In Lung Adenocarcinoma A549 Cells And Transplanted Tumor In Nude Mice

Objective:1.To investigate the effect of knockdown CXCR4 on EGFR expression in A549 cells.2.To investigate the effect of EGF combined with PI3 K inhibitor(LY294002)on CXCR4 expression in A549 cells and the growth of transplanted tumors in nude mice.Methods:1 Design and synthesis CXCR4 siRNA vector,which was transfected into A549 cells.Transfection efficiency was evaluated by qRT-PCR.2 The siRNA with the highest transfection efficiency was transfected into A549 cells.The relative expression of EGFR was detected by qRT-PCR.The expression of EGFR protein was detected by Western-Blot.3 The A549 cells were divided into control group,2?g/mlCXCL12 group,4?g/mlCXCL12 group,then,the cells were treated with 0?g/ml,2?g/ml,or4?g/ml CXCL12 for 24 h,the expression of EGFR protein was detected by Western-Blot.4 The A549 cells were divided into control group,10ng/ml EGF group,40ng/ml EGF group,100ng/ml EGF group,then,the cells were treated with 0ng/ml,10ng/ml,or 40ng/ml,100ng/ml EGF for 24 h,the expression of CXCR4 protein was detected by Western-Blot,to find the optimal concentration for promoting the expression of CXCR4 protein as a subsequent experimental concentration.5 The A549 cells were divided into control group,EGF group,EGF+LY294002 group,LY294002 group,then,the cells were treated with0ng/ml,40ng/ml EGF,20?mol/L LY294002+40ng/mlEGF,20?mol/L LY294002 for 24h,the expression of CXCR4 protein was detected by Western-Blot.6 The invasion ability of four groups in control group,EGF group,EGF+LY294002(PI3K inhibitor)group,LY294002(PI3K inhibitor)group were detected by Transwell chamber.7 The transplanted tumor model of nude mice were established and randomly divided into 4 groups:control group,EGF group,EGF+LY294002group,LY294002 group,the changes in dietary and mental status of nude mice were recorded during treatment.The body weight and tumor volume of nude mice were measured when the completion of treatment and then nude mice were killed.The tumors were excised and the tumor volume was measured,the morphological changes of the tumor cells were observed by HE staining.8 The expression of CXCR4 protein was detected in transplanted tumor of nude mice by Western-Blot and immunohistochemistry.Results:1.The effect of CXCL12/CXCR4 on EGFR expression1.1 The effect of siRNA interfering with CXCR4 on EGFR expression1)A549 cells knocked down by CXCR4 gene were successfully contructed.The results of qRT-PCR was showed that the relative expression of CXCR4 in the transfection group was significantly lower than that in the control group.The results of Western-Blot was showed that the expression of CXCR4 protein in the transfection group was significantly lower than that in the control group.The relative expression level of siRNA2 was the lowest,and the knockdown efficiency was the highest.1.2 The effect of CXCL12 on EGFR expressionThe CXCR4siRNA2 was transfected into A549 cells.The results of qRT-PCR and Western-Blot showed that the expression of EGFR mRNA and protein was significantly reduced.2.The effect of EGFR pathway on CXCR4 expressionA549 cells were treated with different concentrations of CXCL12,Western-Blot showed that the expression of EGFR protein had a dosedependent relationship with CXCL12,that is,the higher CXCL12 concentration,the stronger EGFR expression was.2.1 The effect of EGF on CXCR4 protein expressionA549 cells were treated with different concentrations of EGF,Western-Blot showed that the expression of CXCR4 protein had a dose-dependent relationship with EGF,that is,the higher the EGF concentration,the stonger EGFR expression was.2.2 The effect of PI3 K inhibitor LY294002 and agonist EGF on the expression of CXCR4In the control group,EGF group,EGF+LY294002 group,and LY294 002 group,Western-Blot results showed that the expression of CXCR4 protein was the strongest in the EGF group,the weakest in the LY294002 group,and the EGF+LY294002 group was stronger than that in the LY294002 group.2.3 The cell invasion ability was detected by Transwell chamberThe invasion ability of control group,EGF group,EGF+LY294002group,LY294002 group was detected by transwell chamber experiment,which showed that the EGF group was the strongest,the LY294002 group was the weakest,and the EGF+LY294002 group was stronger than that in single LY294002 group.3.Observing the tumor growth of transplanted tumor in nude mice and its effect on the expression of CXCR43.1 16 nude mice were tumors.There were no significant differences in the diet and mental status during treatment.There was no significant difference in body weight before and after treatment.The tumor volumes were significantly different from that of the control group during treatment.3.2 The HE stained sections under microscope showed that the tumor tissues of xenografts in nude mice were lung adenocarcinoma.The cancer cells had obvious heteromorphism,deep nuclear staining,increased nuclear/plasma ratio,and various cell morphology,the nuclear chromatin was coarse and granular..3.3 The results of Western-Blot showed that,the LY294002 group was the weakest,the EGF group was the strongest,and the EGF+LY294002 group was stronger than that in LY294002 group.3.4 The results of Immunohistochemistry showed that,the LY294002 group was the weakest,the EGF group was the strongest,and the EGF+LY294002group was stronger than that in LY294002 group.Conclusion:1.The CXCR4 siRNA is used in the experiment can obviously knock down the expression of CXCR4 in A549 cells and reduces the expression of EGFR.2.The CXCL12 promotes the EGFR expression through the CXCL12/CXCR4 axis,and plays a positive feedback role.3.The EGF upregulates the expression of CXCR4 and enhances cell invasion ability through the PI3K/AKT signaling pathway in A549 cells and nude mouse transplanted tumors,and then affects the growth function of transplanted tumor.