| Objective: Because of its safety and effectiveness,molecular targeted therapy has been used in the clinical treatment of many tumors including Non small cell lung cancer(NSCLC).Gefitinib is the first approved epidermal growth factor receptor tyrosine kinase inhibitor,which has achieved good therapeutic effect in clinical application,and has become the first-line treatment for advanced NSCLC patients with EGFR gene mutation.However,most patients will be resistant to gefitinib after a period of time.It is important to elucidate the mechanism of gefitinib resistance in NSCLC.MicroRNAs can participate in the regulation of almost all physiological or pathological biological functions by silencing their target genes.Recently,many microRNAs have been found to be involved in the production and maintenance of gefitinib resistance in NSCLC.The purpose of this study is to construct the microRNA-target regulatory network of gefitinib resistant by analyzing the differential expression of gefitinib resistant microRNA in GEO database,and the differentially expressed genes were analyzed by the GO function annotation and KEGG enrichment analysis.The role of miR-342-3p in the formation and regulation of gefitinib resistance in NSCLC cells and the related molecular mechanism were clarified,so as to provide a new basis for elucidating the mechanism of NSCLC molecular target therapy.Methods: 1.Data sets of NSCLC gene expression profiles were obtained from GEO database for integration and analysis,and differential expression genes were identified.2.The differentially expressed genes were analyzed by GO function annotation and KEGG enrichment analysis.3.To construct the gene resistant microRNA-target regulatory network of NSCLC.4.Culture of A549 and PC9 cells and gefitinib resistant A549/GR and PC9/GR cells.5.Real-time quantitative PCR was applied to examine the expression of miR-342-3p and CPA4 mRNA.6.MicroRNAs or plasmids were transfected into A549/GR and PC9/GR cells.7.The cell viability and gefitinib sensitivity of A549/GR and PC9/GR cells was detected using CCK8 assay.8.The regulation of miR-342-3p on CPA4 gene was detected by luciferase reporter gene experiment.9.The expression of CPA4 protein was examine by Western blotting.Results: 1.Based on the data set of GEO database,the differentially expressed genes ofgefitinib resistance in NSCLC were identified.2.Construct the microRNA-target regulatory network of gefitinib resistance.3.The differentially expressed genes waer analyzed by GO function annotation and KEGG enrichment analysis.4.The expression of miR-342-3p in drug resistant A549/GR and PC9/GR cells was significantly higher than that in their A549 and PC9 cells(P<0.05).5.The overexpression of miR-342-3p can reduce the gefitinib sensitivity of A549/GR and PC9/GR cells and enhance the gefitinib resistance(P<0.05);the silencing of miR-342-3p can significantly improve the gefitinib sensitivity of A549/GR and PC9/GR cells and inhibit the gefitinib resistance(P<0.05).6.MiR-342-3p can negatively regulate the expression of CPA4 m RNA and protein(P<0.05).7.Luciferase reporter gene experiment showed that miR-342-3p could target the 3'UTR region of CPA4 gene(P<0.05).8.Compared with A549/GR and PC9/GR cells in miR-342-3p mimic + pC-NC group,A549/GR and PC9/GR cells in miR-342-3p mimic + pC-CPA4 group up-regulated the expression of CPA4 protein(P<0.05).9.Compared with A549/GR and PC9/GR cells in miR-342-3p mimic + pC-NC group,the gefitinib sensitivity of A549/GR and PC9/GR cells in miR-342-3p mic + pc-CPA4 group was significantly increased,and the gefitinib resistance was decreased(P<0.05).Conclusion: The differentially expressed genes of gefitinib resistance in NSCLC were identified,the microRNA-target regulatory network of gefitinib resistance in NSCLC was constructed,and GO function annotation and KEGG enrichment analysis were carried out for the differentially expressed genes.Mir-342-3p can reduce the gefitinib resistance of NSCLC cells by silencing its target gene CPA4. |
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