Bioinformatics-Based Gene Analysis Of Gefitinib Resistance In NSCLC And Mechanism Investigation Of Serine Protease 8 Involved In Gefitinib Resistance In NSCLC

The epidermal growth factor receptor(EGFR)is critical in the incidence and progression of non-small cell lung cancer(NSCLC).According to research,EGFR is extensively expressed on the surface of NSCLC cells and is frequently accompanied by mutations,the most common of which are exon 19 deletion and exon 21 L858 R point mutations.Targeted therapy targeting the EGFR gene is considered effective in the treatment of NSCLC.By targeting EGFR,epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)significantly increase median progression-free survival in patients with EGFR mutations.Gefitinib is one of the representative drugs of EGFR-TKIs.Unfortunately,after receiving EGFR-TKIs for 10-14 months,patients usually develop acquired resistance to them.It remains a major concern for treating NSCLC that EGFR-TKI resistance mechanisms are unpredictable and diverse.Part Ⅰ Discovery of gefitinib resistance-associated genes and their mechanisms of action in non-small cell lung cancer based on bioinformaticsObjective: Utilizing bioinformatics to investigate gefitinib resistance hub genes and their role in signaling pathways in NSCLC,and verifying the expression levels of these genes at the cellular level.Methods: 1.Gene expression profiles of GSE122005 and GSE123066 were downloaded from the Gene-on-a-chip database(GEO).Both datasets contained gefitinib-sensitive and resistant NSCLC cell lines.The gene expression profile of gefitinib-resistant and gefitinib-sensitive NSCLC cells was compared using the R package “limma”.2.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genom)enrichment analysis of the DEGs in the two datasets,respectively.3.The intersection of the differential genes up-regulated and down-regulated in the two datasets was taken separately.And the key genes involved in gefitinib resistance were screened through the protein-protein interaction(PPI)network.4.The expression levels of hub genes in lung cancer were first verified using the GEPIA(Gene Expression Profiling Interactive Analysis)database.5.The expression levels of hub genes in NSCLC gefitinib-resistant cell lines(PC9/GR)and sensitive cell lines(PC9)were assessed using quantitative real-time PCR(qRT-PCR).Results:1.From the GSE122005 dataset,we identified 2553 differential genes(346 genes were up-regulated and 1207 genes were down-regulated in resistant strains).In the GSE123066 dataset,327 genes were highly expressed in resistant cell lines and 479 genes were lowly expressed in resistant cell lines based on a different analysis of 6 samples.2.According to the enrichment analysis,genes associated with gefitinib resistance in NSCLC have been significantly enriched in the PI3K/AKT and MAPK signaling pathways.3.It was found in this study that 43 genes were co-up-regulated and 86 genes were co-down-regulated from the intersection of differential genes in GSE122005 and GSE123066.The top 20 key genes related to gefitinib resistance in NSCLC screened by constructing a PPI network were MAL2 、 ST14、 PRSS8、 F11R、CGN、MARVELD3、S100A14、RAB25、FOXA2、DKK1、CSF2、IGFBP3、 LOX、 TGM2 、 PTGS2、 PLAU、 CYP1A1、 MUC20、PVL、 CDH3.4.The GEPIA database results revealed that the expression levels of four hub genes associated with gefitinib resistance in non-small cell lung cancer,MAL2,ST14,PRSS8,and F11 R,were significantly higher in lung adenocarcinoma than in normal lung tissue.5.According to the qRT-PCR results,PRSS8 and MAL2 expressed significantly more in PC9/GR cells than in PC9 cells(P<0.0001).In comparison to PC9 cells,ST14 expression was considerably lower in PC9/GR cells(P<0.01).A significant difference was not observed between PC9/GR and PC9 cells in terms of F11 R expression.Conclusions: It has been discovered that signaling pathways(PI3K/AKT)and hub genes(PRSS8、MAL2 and ST14)could contribute to NSCLC resistance to gefitinib.These results may be useful in studying the mechanism of gefitinib resistance in NSCLC and may provide a theoretical basis for subsequent experimental studies.Part Ⅱ Mechanisms of serine protease 8 involvement in gefitinib resistance in non-small cell lung cancerObjective: To investigate the expression level of serine protease 8(PRSS8)in non-small cell lung cancer gefitinib-resistant cell lines(PC9/GR)and its mechanism of action.Method: The expression levels of PRSS8 in gefitinib-resistant cell lines(PC9/GR)and sensitive cell lines(PC9)of NSCLC were detected by Western blot.It was constructed and transiently transferred into PC9/GR cells the small interfering RNA of PRSS8(PRSS8-siRNA),which only silences PRSS8 gene expression in PC9/GR cells.The study was divided into the transfected PRSS8-siRNA PC9/GR cell group and the Con-siRNA PC9/GR cell group.Western Blot was used to detect PRSS8 gene expression in PC9/GR cells after PRSS8 gene expression was knocked down.Furthermore,after PRSS8 expression was silenced,alterations in downstream signaling pathway components and phenotypic protein expression were observed in PC9/GR cells.We transfected PC9/GR cells with PRSS8-siRNA and Con-siRNA and cultured them with 800ng/ml gefitinib.The proliferation of PC9/GR cells at 24 、 48 、 and 72 h was detected by CCK8.Simultaneously,a PRSS8 recombinant plasmid was created and transfected into PC9/GR cells.The research was divided into the PC9/GR plasmid group and the no-load group.The expression of the PRSS8 gene and downstream signaling pathway molecules in PC9/GR cells from the two groups was detected using Western Blot.Results: Western blot results showed that the expression of PRSS8 in PC9/GR cells was significantly higher than that in PC9 cells,and the difference was statistically significant(P<0.05).It was observed that the expression of PRSS8 in transfected PRSS8-siRNA cells was significantly lower than that in the Con-siRNA group in PC9/GR cells(P<0.05).The expression of PRSS8,p-AKT,p-m TOR,β-catenin,and BCL-2 proteins was significantly reduced in the PRSS8-siRNA cell group compared to the Con-siRNA cell group.There was a significant increase in Caspase9 and Bax expression in the Con-siRNA PC9/GR group(P<0.05).The proliferative activity of the PRSS8-siRNA cell group after gefitinib treatment was significantly lower than that of the Con-siRNA cell group at 24、48、and 72 hours.In PC9/GR cells,the expressions of PRSS8,p-AKT,p-m TOR,β-catenin,and BCL-2 proteins were significantly increased in the plasmid group and the expression of Bax was reduced considerably in the plasmid group compared to the no-load group,with statistically significant differences.Conclusion: PRSS8 may play a causal role in the emergence of acquired gefitinib resistance in non-small cell lung cancer by regulating the AKT/m TOR and β-catenin signaling pathways.