The Effect Of Epidermal Growth Factor Receptor On CXC Chemokine Receptor 4 Expression In Invasion And Metastasis Of Lung Adenocarcinoma

Part one The effects of EGFR and downstream ERK pathway on CXC-R4 expression in vivo and in vitroObjective: We investigate the influence of EGFR on the expression of CXCR4 by siRNA interference which can silence the expression of EGFR in lung adenocarcinoma cells(A549 cells).We apply the ERK inhibitor U-0126 and ERK agonist EGF in A549 cells and transplanted tumor in nude mice to explore the expression level of CXCR4 and the invasion ability of A549 cells.Methods:1 After transfecting EGFR siRNA which could slience the EGFR gene,Real Time-PCR and Western-Blot were used to detect the mRNA and protein expression of EGFR.According to the result,the siRNA with the best silencing effect was selected for subsequent experiments.Then the trail was divided into two groups: experimental group(EGFRsiRNA),Control group(ControlsiRNA).Real Time RT-PCR and Western-Blot were used to detect changes in expression level of CXCR4.2 We stimulated A549 cells with different concentration of EGF(10、40and100 ng/mL)for 24 h and examined the changes in expression of CXCR4 by Western-Blot to investigate whether CXCR4 showed marked increase in concentration dependent manner.3 The A549 cells in the logarithmic growth phase were digested.The trial was divided into four groups: untreated group(without any drug intervention),EGF group,EGF + U-0126 group,U-0126 group,which were incubated for 24 h.The expression of CXCR4 protein in A549 cell was detected by Western Blot.4 The invasion ability of A549 cells in each group was detected bytranswell chambers.Migration cell number was counted to evaluate the invasion ablilty of A549 cells in each group.5 The A549 cells were injected to right axilla subcutaneously of nude mice using syringe.16 nude mice were inoculated and feeded about 15 days.16 nude mice transplanted with A549 cells were randomly divided into 4groups.Those were untreated group,EGF group,EGF + U-0126 group and U-0126 group.The diet and the mental state of nude mice were recorded during the course of medication,besides the body weight and tumor size were measured.All the nude mice were put to death after medication.We stripped tumors completely,measured tumor size and observed morphological changes by HE staining.6 The expression level of CXCR4 protein in transplanted tumor of nude mice was detected by Western Blot and immunohistochemical method.Result:1.The silencing effect of specific EGFRsiRNA on EGFR expression and the expression of CXCR4 after silencing EGFR.After treatment of lung adenocarcinoma A549 cells with the three different EGFR siRNAs,the mRNA and protein expression levels of EGFR were significantly reduced,and the maximum silencing efficiency one was the EGFRsiRNA-1.After the expression of EGFRsiRNA-1 silencing EGFR,Real Time-PCR,Western-Blot results showed that the expression level of CXCR4 was remarkably reduced by siRNA-mediated knockdown of EGFR in A549 cells.2.The expression of CXCR4 is dependent on the concentration of EGF.Through the disposal of EGF in different concentrations,the expression of CXCR4 protein presented concentration dependent manner by Western-Blot.3.The effects of ERK inhibitor U-0126 and agonist EGF on expression of CXCR4 protein in A549 cells.The A549 cells in the logarithmic growth phase were divided into four groups,which were treated with different drugs for 24 h.The expression ofCXCR4 protein was detected by Western-Blot.By contrast with control group,the expression of CXCR4 remarkably increased in EGF group.On the contrary,the expression of CXCR4 protein in the EGF+U-0126 group and U-0126 group were significantly declined compared with the control group and EGF group.4.The invasion ability of A549 cells was detected by the Transwell chamber.We observed that the migration cell number of A549 cells in EGF group was dramatically more than the control group.It could be seen that EGF can promote the invasion ability of A549 cells.The migration cell number of A549 cells in EGF+U-0126 group and U-0126 group were significantly less than EGF group and untreated group.It could be concluded that U-0126 could restrain the invasion ability of A549 cells.5.The changes and morphological observations of the tumor volume were observed in the nude mice of each group.Tumor formation rate of 16 nude mice is 100%.Significant differences of the tumor size were found in each group(P<0.05)..Morphological observation results in nude mice orthotopic transplanted tumor in each group:The tumor tissues of nude mice transplanted tumor were lung adenocarcinoma.Cell morphological were various.The nuclear chromatins showed coarse granular and nucleolus were obvious.6.Immunohistochemistry and Western-Blot were used to detect the effects of different drugs on the expression of CXCR4 in nude mice with lung adenocarcinoma.The expression of CXCR4 protein was detected by Western-Blot and immunoreactive score.By contrast with untreated group,EGF remarkably increased the expression of CXCR4 in EGF group.On the contrary,the expression of CXCR4 protein in the EGF+U-0126 group and U-0126 group were significantly declined compared with the untreated group and EGF group.Part two The effect of EGFR mutation status on the invasion,metast-asis biological behaviors in lung adenocarcinomaObjective: The differences in expression of CXCR4,Ki67,MDM2 and N-Cadherin are studied to explore the relationship between EGFR mutation status and tumor biological characteristics by immunocytochemical stain-ing.We hope to offer some new sources of inspiration for clinic research trials and treatments.Methods:1 Pleural effusion of lung carcinoma patients were collected by centrifugation to obtain cell sediment in Fourth Hospital of Hebei Medical Univercity from July 2017 to December 2017.2 The initial screening and diagnosis of pleural effusion cells were performed by using hematoxylin-eosin staining(HE staining).3 Immunocytochemistry(ICC)was used to identify the properties and types of pleural effusion cells.4 The EGFR gene of pleural effusion cells was detected by amplifi-cation refractory mutation system(ARMS),which could rapidly and sensitively screen for the EGFR mutation.5 The expression of CXCR4,Ki67,MDM2,N-Cadherin were detected by immunocytochemistry(ICC)to explore the differences in expression of them between the EGFR sensitive mutation group and non-mutation group.Result:1.HE staining was used to screen the pleural effusion cells.HE staining was diagnosed by the two cytopathologists.It could be seen that there were abundant malignant tumor cells,various cell types,deep-dyed big nucleolus,coarse-grained chromatin and increased nucleoplasmic ratio which was conformed to requirements of further immunocytochemical staining and ARMS detection.2.Immunocytochemical staining was used to further determine the properties and types of pleural effusion cells.The antibody TTF-1 and NapsinA in the cytological smear of lungadenocarcinoma were used for immunocytochemical staining,both of that were positive,which could be further diagnosed as lung adenocarcinoma.In addition,wt-1 and Calretinin,ck5/6,p63,CD56 and Syn were added for identification3.ARMS method was used to detect EGFR mutations in pleural effusion.Amplification refractory mutation system PCR(ARMS-PCR)indicated31 EGFR sensitive mutation cases and 15 EGFR non-mutation cases among46 pleural effusion cases of lung adenocarcinoma.4.The expression of CXCR4,MDM2,Ki67 and N-Cadherin in the two groups were detected by immunocytochemistry.Immunocytochemical staining indicated that the expression of CXCR4,Ki67,MDM2,N-Cadherin had significant relationship with the EGFR mutation status.Significant statistical differences in the expression of CXCR4,Ki67,MDM2,N-Cadherin were showed between the EGFR sensitive mutation group and non-mutation group.Conclusions:1.The ERK pathway inhibitor U-0126 could restrain the expression of CXCR4 protein induced by EGF to reduce the invasion ability of A549 cells.Inhibitor U-0126 could also suppress transplanted tumor growth in nude mice.2.The mutation status of pleural effusion cells was related to the biological behaviors such as proliferation,invasion,metastasis of lung adenocarcinoma.