ANGPTL4 Regulates Lung Adenocarcinoma Cell Apoptosis Through ERK1/2 Pathway And Affects Gefitinib Resistance

Objective Use sh RNA to transfect gefitinib-resistant lung adenocarcinoma cells PC9/GR to interfere with the expression of ANGPTL4 in PC9/GR cells,observe the effect of PC9/GR on gefitinib resistance after interfering with ANGPTL4 and explore its related mechanisms.Method1.Use the TCGA database to analyze the expression of ANGPTL4 in pan-carcinoma;then use the GEPIA database to analyze the difference in the distribution of ANGPTL4 in lung adenocarcinoma tissues and normal tissues,and the difference in expression of ANGPTL4 in patients with lung adenocarcinoma of different stages;use the TCGA database,GSE The database downloads the survival status of lung adenocarcinoma patients with high and low expression of ANGPTL4,and draws the survival curve.2.Culture human lung adenocarcinoma cells PC9 and gefitinib-resistant cells PC9/GR,and use q RT-PCR and Western blot methods to detect the m RNA and protein expression levels of ANGPTL4 in the two groups of cells.3.Construct ANGPTL4-sh RNA and transfect PC9/GR cells to interfere with the expression of ANGPTL4.Western blot method was used to detect the interference efficiency.4.Four groups of PC9,PC9/GR,PC9/GR-sh NC,PC9/GR-sh ANGPTL4 were selected,and the cells were treated with media supplemented with different concentrations of gefitinib for 24 hours,and then the IC50 was detected by CCK-8,To explore the effect of ANGPTL4 expression level on the drug resistance of PC9/GR cells.5.Culture the three groups of PC9/GR,PC9/GR-sh NC,PC9/GR-sh ANGPTL4 cells,and add gefitinib to culture.EDU experiment,flow cytometry and Transwell experiment were used to detect the proliferation activity,apoptosis level and invasion level of each group of cells,and the correlation between ANGPTL4 and PC9/GR cell growth and invasion ability was clarified.6.Use PC9/GR cells and interfering strains to construct a nude mouse xenograft model.After grouping,they were fed with PBS and gefitinib and the changes in the body weight and tumor tissue volume of the mice were recorded.7.Peel the transplanted tumor tissue and perform Ki-67 immunohistochemistry and Tunel staining,to clarify the influence of ANGPTL4 on the invasion ability and apoptosis level of PC9/GR cells in the tissue.8.Use PC9/GR cells and interfering strains to construct a nude mouse xenograft model.After grouping,they were fed with PBS and gefitinib and the changes in the body weight and tumor tissue volume of the mice were recorded.9.Extract the proteins of the above 7 groups of cells and the proteins in the tumor tissues of the four groups of transplanted tumor animal models,and use Western blot to detect ERK1/2,BAX,Bcl-2,caspase 8,cleaved-caspase 8 in each group of samples The expression level of apoptosis-related proteins is based on the upstream and downstream regulatory relationship between ANGPTL4 and ERK1/2 apoptosis pathway at the cell and animal level,under the condition of interference and overexpression.Results1.Download the patient information in the TCGA,GEPIA,GSEO database,and confirm that ANGPTL4 is elevated in a variety of malignant tumor tissues,and is significantly negatively correlated with the patient’s prognosis（p<0.05）.2.Both q RT-PCR and Western blot experiments found that PC9/GR cells have higher ANGPTL4 expression levels than PC9 cells（p<0.01）.3.Compared with PC9,PC9/GR has a higher IC50,confirming that PC9/GR is less sensitive to gefitinib than PC9,and interference with ANGPTL4 can increase the drug sensitivity of gefitinib（p<0.01）.4.Compared with the con group,the cells of the G group have lower proliferation activity and invasion ability,while the level of apoptosis increased;and the sh-ANGPTL4 group has lower proliferation activity and invasion ability,and the level of apoptosis compared with the G group cells Significant increase（p<0.05）.5.Nude mice in the con+G group,sh-NC+G group,sh-ANGPTL4 group,and sh-ANGPTL4+G group showed no significant difference in body weight growth,but the tumor volume decreased sequentially（p<0.05）.6.Compared with the con+G group,the expression level of Ki-67 and the number of apoptotic bodies in the tumor tissues of the sh-NC+G group did not change significantly,while the expression level of Ki-67 and apoptosis in the tumor tissues of the sh-ANGPTL4 group The number of bodies was higher than that in the con+G group（p<0.01）,and the difference was more significant in the sh-ANGPTL4+G group（p<0.001）.7.Western blot experiment proved: p-ERK1/2 expression decreased in PC9/GR cell lines and transplanted tumor tissues that interfered with ANGPTL4 expression,while apoptotic protein increased;after over-expression of ANGPTL4 in PC9/GR cells,p-ERK1 /2 increases the expression,and the expression level of apoptotic protein decreases accordingly.Conclusion ANGPTL4 can inhibit the apoptosis of lung adenocarcinoma PC9/GR cells by regulating the ERK1/2 pathway,thereby promoting the generation of gefitinib resistance.