| ObjectiveEpidermal growth factor receptor(EGFR)exon 19 deletion or exon 21 mutation occurs in approximately 40%of patients with lung adenocarcinoma.Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)have resulted in a significant survival benefit for patients with EGFR-mutated lung adenocarcinoma.However,patient response to EGFR-TKIs is usually transient,as tumors eventually develop resistance to EGFR-TKIs through on-targeted or off-targeted mechanisms.Numerous clinical studies and their meta-analyses have shown that the combination of EGFR-TKIs with traditional Chinese medicine(TCM)significantly improves the overall treatment efficiency,patient quality of life and one-year survival rate in patients with non-small cell lung cancer(NSCLC)compared to EGFR-TKIs alone.Yi-Fei San-Jie pill(YFSJP)is an empirical Chinese herbal formula for lung cancer,which was established by Professor Lin Li-Zhu,a famous Chinese medical oncologist.In our group’s previous clinical study,we found that in advanced EGFR mutation-positive NSCLC patients,TCM treatment with YFSJP as the main formula combined with EGFR-TKIs could extend their median progression-free survival to 11.8 months(better than 5.8-10.8 months with EGFR-TKIs alone)and reduce the risk of disease progression.In addition,biological experiments revealed that YFSJP could inhibit the proliferation of EGFR-TKIs resistant cell lines,increase the sensitivity of resistant cells to EGFR-TKIs,and block the cell cycle by downregulating the PI3K/Akt/mTOR pathway and upregulating autophagy,and the upstream molecular mechanisms of EGFR-TKIs resistance are yet to be explored.In this study,we aimed to investigate the role of YFSJP in inhibiting NSCLC and interfering with EGFR-TKIs resistance,and to predict the specific molecular mechanism of YFSJP interfering with EGFR-TKIs resistance based on multi-omics and bioinformatics,to clarify the effect of miR-335-5p and MET on EGFR-TKIs resistance in lung cancer,and to evaluate the effect of miR-335-5p and MET on EGFR-TKIs resistance.drug resistance,evaluate the therapeutic value of miRNA-335-5p/MET/EGFR-targeted modulation on EGFR-TKIs therapeutic sensitization and improvement of clinical prognosis in lung cancer,so as to elucidate the mechanism of YFSJP for the treatment of NSCLC,and provide theoretical support and experimental proof for the scientific intervention of TCM in acquired drug resistance in mid-to late-stage lung cancer.Methods1.YFSJP reverses NSCLC EGFR-TKIs resistance in vitro and in vivoUsing human lung adenocarcinoma EGFR-TKIs-sensitive cell line PC-9,EGFR-TKIsresistant cell line H1975 and BALB/C nude mouse H1975 cell line subcutaneously transplanted tumors as models,experiments were carried out in vitro and in vivo.At the cellular level,the effects of YFSJP in synergy with EGFR-TKIs on NSCLC cell proliferation,P-gp activity,cell cycle and cell migration were observed by CCK8 assay,Rho123 accumulation assay,flow cytometry cell cycle assay,and Transwell cell migration assay,respectively.The effect of YFSJP on the body weight of tumor-bearing nude mice,the inhibition of tumor growth in synergy with EGFR-TKIs and its safety were observed in vivo.2.Integrating network pharmacology and metabolomics to predict the mechanism of YFSJP on EGFR-TKIs resistance in NSCLCFirstly,blank rat serum and drug-containing serum of YFSJP were prepared in advance.Based on ultra performance liquid chromatography coupled to quadrupole timeof-flight mass/mass spectrometry(UPLC-Q/TOF-MS/MS),the metabolic profiles of rat blank serum and drug-containing serum of YFSJP were obtained.Unmonitored principal component analysis(PCA),Orthogonal partial least Square discriminant analysis(OPLSDA),and literature search were used to analyze the differential metabolites between the two groups.SwissTargetPrediction,Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),PharmMapper and other drug target databases were applied to search for molecular targets of the active components of YFSJP.GeneCards database were applied to screened NSCLC related gene targets.Using Search Tool for the Retrieval of Intervening Genes(STRING)database,a proteinprotein interaction(PPI)network was constructed for the intersection targets of the molecular targets of the active components of YFSJP and NSCLC related gene targets.The"YFSJP-component-target-NSCLC" network was constructed by Cytoscape 3.8.0 software.Gene ontology(GO)analysis and Kyoto encyclopedia of gene and genome enrichment(KEGG)pathway analysis were performed on the YFSJP core targets using the clusterProfiler R package.Finally,the dataset of EGFR-TKIs resistance from National Center for Biotechnology Information(NCBI)Gene Expression Omnibus(GEO)and the dataset of lung adenocarcinoma(LUAD)from The Cancer Genome Atlas(TCGA)were used to perform the analysis of gene differential expression.The mRNA-miRNA regulatory network of LUAD EGFR-TKIs drug resistance regulated by YFSJP was constructed by intersection with the core target of YFSJP and the differential genes of LUAD EGFR-TKIs resistance.3.YFSJP reverses NSCLC EGFR-TKIs resistance by modulating MET-associated miRNA-335-5pYFSJP combined with EGFR-TKIs were used to treat EGFR-TKIS sensitive or drugresistant NSCLC in vitro and in vivo.Real-time quantitative Polymerase Chain Reaction(RT-qPCR),Western blot(WB)and Immunohistochemistry(IHC)were performed to observe the changes of miR-335-5p and MET molecules at the level of transcription and protein.To verify the prediction results of network pharmacology,subsequently,we analyzed the direct regulatory relationship between miR-335-5p and MET by dual luciferase reporting assay.Then lentiviral NSCLC stable strains with low or overexpression of miR-335-5p were constructed to observe the changes in cell proliferation,drug storage capacity,cell cycle and tumor migration of the two stable strains.Finally,the expression changes of miR-335-5p and MET molecules at the transcriptional level and protein level were observed by RT-qPCR,WB and IHC in vivo and in vitro.Results1.YFSJP reverses NSCLC EGFR-TKIs resistance in vitro and in vivoIn vitro,CCK8 showed that Gef had a significant inhibitory effect on PC-9 cells compared with H1975 cells,and H1975 cells were resistant to gefitinib(Gef).The IC50 of PC-9 cells and H1975 cells were 0.218μM and 41.641μM respectively after Gef treatment for 48h.In both cell lines,the combination of different doses of Gef and 15%YFSJPcontaining serum significantly reduced cell viability compared to monotherapy with Gef.The results of Rho123 accumulation experiment showed that the H1975 cell line in negative control(NC)group had a lower accumulation level of Rho123 compared with PC9 cells in NC group,suggesting that the drug resistance level of H1975 to Gef was significantly higher than that of PC-9.After treated with Gef+YFSJP containing serum for 48h,Rho123 accumulation in PC-9 and H1975 cell lines was significantly higher than that in Gef group.Compared with NC group,the percentage of G2 phase cells in Gef group,YFSJP group and combination group was significantly reduced,and Gef and YFSJP could induce cell arrest in G1/S phase.Compared with the Gef group,the percentage of G2 phase cells in the Gef+YFSJP combined group was significantly reduced,indicating that the combined treatment could enhance the effect of Gef on inducing NSCLC cell cycle arrest in G1/S phase.Transwell results showed that the number of cells migrating to the lower compartment of both strains was significantly reduced compared with the NC group 24 h after drug treatment.In addition,the number of migrating cells in PC-9 and H1975 cells in Gef+YFSJP-containing serum combination group was also significantly reduced compared with the Gef monotherapy group.In vivo,the subcutaneous transplantation model of BALB/C nude mice was successfully constructed with H1975 cell line.The model was divided into negative control(NC)group,Gef group,YFSJP group,Gef+YFSJP group,Normal saline,Gef(50mg/kg/d),YFSJP(9g/kg/d)and Gef(50mg/kg/d)combined with YFSJP(9g/kg/d)were given for 21 days,respectively.The tumor size and weight of Gef group,YFSJP group and Gef+YFSJP group were all smaller than those of NC group,and Gef+YFSJP group was smaller than the other two groups.During drug intragastric administration,there was no statistical difference in the weight of tumor-bearing nude mice in each group,but the weight loss rate of YFSJP group and Gef+YFSJP group was slower than that of other groups.The results of tumor volume curve in tumor bearing nude mice showed that NC group had the fastest tumor volume increase,while Gef+YFSJP group had the slowest tumor volume increase.The results of serum liver and kidney function test in tumor-bearing nude mice showed that there was no significant difference between NC group,Gef group,YFSJP group and combination group.The organ weight index of heart,liver,spleen,lung and kidney in nude mice with tumor showed no significant difference among the four groups.2.Integrating network pharmacology and metabolomics to predict the mechanism of YFSJP on EGFR-TKIs resistance in NSCLCIn the metabolomics study of YFSJP,UPLC-Q/TOF-MS/MS technology was used to compare the metabolic characteristics of YFSJP rat drug-containing serum samples and rat blank serum samples in positive and negative ion mode.A total of 118 different metabolites were identified,and YFSJP was involved in 18 amino acid metabolic pathways.The tyrosine metabolic pathway had the highest peak.Using SwissTargetPrediction,TCMSP and other drug target databases,we obtained 667 target genes of 30 antitumor active components of YFSJP,and 4684 NSCLC disease target genes were retrieved from GeneCards database.A total of 358 intersection target genes between YFSJP and NSCLC were obtained to construct the "YFSJP-componenttarget-NSCLC" network.The GO enrichment results of 358 target genes showed that the regulation of YFSJP in NSCLC was mainly related to biological processes such as adenylate cyclase and cyclic nucleotide second mRNA mediated G-protein-coupled receptor signaling pathway and positive regulation of protein serine/threonine kinase activity.KEGG pathway analysis results were closely related to the regulation of NSCLC by YFSJP,including EGFR-TKIs resistance,chemical carcinogenesis,PI3K-Akt signaling pathway,PD-L1 expression in cancer and PD-1 checkpoint pathway,proteoglycan in cancer,tumor necrosis factor signaling pathway,apoptosis,and T cell receptor signaling pathway,platinum drug resistance pathway,etc.The 358 selected intersection target genes were imported into STRING network platform to obtain STRING-PPI network,which contained 86 key proteins.The plug-ins of Cytoscape 3.8.0 software BisoGenet and CytoNCA were used to analyze the network centrality topology of these 86 key proteins,and the Cytoscape-PPI network of 86 key proteins was constructed.Finally,a total of 27 core targets of YFSJP for NSCLC were obtained.R software was used to analyze gene expression differences in EGFR-TKIs drug-resistance related dataset GSE63678 and TCGA-LUAD queue RNA-seq dataset.As a result,2044 EGFR-TKIs drug-resistance related genes and 2657 LUAD related genes were identified.The intersection of the two with the target of YFSJP has obtained 28 target genes of YFSJP for the intervention of EGFR-TKIs resistance in NSCLC.KEGG signaling pathway enrichment analysis of these 28 target genes showed that YFSJP interfered with EGFR-TKIs drug resistance of lung cancer through PI3K-Akt signaling pathway,EGFR-TKIs drug resistance,microRNA in cancer and other pathways.R software was used to analyze gene expression differences in TCGA-LUAD queue RNA-seq dataset and miRNA-seq dataset.As a result,2653 differential mRNAs and 342 differential miRNAs were identified for the construction of LUAD-related mRNA-miRNA regulatory network.The intersection of 27 core targets of YFSJP in Cytoscape-PPI network for NSCLC treatment,2044 genes related to drug resistance of EGFR-TKIs,and 2653 mRNAs in LUAD-miRNA regulatory network was obtained.The intersection target genes of YFSJP regulating EGFR-TKIs drug-resistant mRNA-miRNA network in NSCLC are ERBB2,MET,ESR1 and EGFR,so as to obtain 15 miRNAs regulating these 4 genes in the mRNA-miRNA regulatory network.And they were used to construct mRNA-miRNA regulatory networks as YFSJP to regulate EGFR-TKIs resistance.Clinical data from the TCGA-LUAD cohort were used for the survival analysis of the above 15 mRNAs.The results showed that miRNA-335-5p was the most significant miRNA with prognostic value,and it was simultaneously regulated by two core target genes,MET and EGFR,in the YFSJP regulation of EGFR-TKIs drug-resistant mRNAmRNA network of NSCLC.3.YFSJP reverses NSCLC EGFR-TKIs resistance by modulating MET-associated miRNA-335-5pRT-qPCR results showed that YFSJP combined with Gef up-regulated miRNA-335-5p and down-regulated the mRNA expression levels of MET and EGFR both in vitro and in vivo.WB,IHC and other experimental results showed that YFSJP combined with Gef may up-regulate miRNA-335-5p and down-regulate p-MET,p-EGFR,CDK2 and CDK4 related proteins of MET/EGFR pathway to intervene in NSCLC cell proliferation,cell cycle arrest,cell migration and EGFR-TKIs resistance.Dual luciferase reporter assay results showed that miR-335-5p directly regulates the expression of MET through the MET 3’UTR binding site.Then,stably blocked and overexpressed miR-335-5p cell lines were constructed by lentivirus transfection,and the expression of miR-335-5p in stably blocked miR-335-5p cell lines was negatively regulated with the mRNA levels of MET and EGFR and MET/EGFR-related pathway proteins.CCK8 assay and Rho123 accumulation assay were used to detect the survival rate and P-gp activity of the cells in each group by blocking and overexpressing miR-335-5p and its control stable strains,respectively.The results showed that up-regulation of miRNA-3355p expression could improve the sensitivity of cells to Gef,which in turn could lead to drug resistance of cells to Gef.Cell cycle experiment results showed that overexpression of miR-335-5p could promote cell cycle arrest in Gl/S phase of NSCLC,and blocking miR335-5p could induce significantly reduced cell cycle arrest in G1/S phase of NSCLC.Transwell tumor migration experiment results showed that overexpression of miR-335-5p inhibited the migration ability of NSCLC cells,while blocking Mir-335-5P significantly enhanced the migration ability of NSCLC cells.In vivo,overexpression of miR-335-5p inhibited the growth of subcutaneous transplanted tumors of NSCLC in nude mice and increased the sensitivity of NSCLC to Gef.Conclusion1.The combination of YFSJP drug-containing serum and Gef can reduce cell viability,drug resistance,cell migration ability and inhibit cell proliferation by inducing cell cycle arrest in G1/S phase of NSCLC cell lines in vitro.In addition,YFSJP combined with Gef can effectively inhibit the growth of subcutaneous xenografts of lung cancer EGFR-TKIs-resistant cell line H1975 in vivo,without additional liver and kidney toxicity compared with Gef or YFSJP monotherapy.2.Metabolomics pathway enrichment analysis showed that the main metabolic signaling pathway of YFSJP was the tyrosine metabolism pathway.The results of network pharmacology GO and KEGG enrichment analysis indicated that YFSJP may treat NSCLC by regulating EGFR-TKIs resistance pathway,miRNA in cancer and other signaling pathways.Combining the analysis results of metabolomics,network pharmacology and bioinformatics,we predicted that the miR-335-5p and MET may be the important mechanism of YFSJP reversing EGFR-TKIs resistance in NSCLC.3.YFSJP combined with Gef may interfere with NSCLC cell proliferation,cell cycle arrest,cell migration and EGFR-TKIs resistance in vitro and in vivo by up-regulating miR-335-5p and down-regulating MET/EGFR pathway.In addition,miR-335-5p plays an important role in indirectly mediating EGFR expression changes by directly regulating MET.Overexpression of miR-335-5p at the post-transcriptional level interferes with NSCLC cell proliferation,cell cycle arrest,cell migration,EGFR-TKIs resistance in vitro and tumorigenesis in vivo. |
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