Study On The Mechanism Of Astragalus Polysaccharide Reversing NSCLC Gefitinib Acquired Resistance Through TLR4 Pathwa

Objective:1.Using bioinformatics research methods,the core genes related to acquired resistance to gefitinib in NSCLC were excavated,and the mechanism of Astragalus Polysaccharide(APS)combined with gefitinib in reversing acquired resistance to gefitinib in NSCLC was preliminarily explored.2.In vitro experiments were used to explore the effects of Astragalus polysaccharides combined with gefitinib on the proliferation,apoptosis,invasion and migration of NSCLC gefitinib acquired resistance,and to further explore the molecular mechanism of Astragalus polysaccharides combined with gefitinib on NSCLC gefitinib acquired resistance,and to clarify the mechanism of Astragalus polysaccharides combined with gefitinib reversing NSCLC gefitinib acquired resistance,so as to provide a basis for subsequent in vivo experiments and clinical applications.Methods:1.Download the mRNA expression profile of human non-small cell lung cancer with acquired gefitinib resistance from the Gene Expression Omnibus(GEO)(https://www.ncbi.nlm.nih.gov/geo/)database,GSE34228 dataset.We selected two groups of untreated samples,including 26 PC9/GR cell samples in the acquired gefitinib-resistant group and 26 PC9 cell samples in the gefitinib-sensitive group,and normalized them.Based on the R language environment,the "Limma" package was used to extract and analyze the differentially expressed data to determine the differentially expressed genes(DEGs).Subsequently,the data were enriched and analyzed by ClusterProfiler in R,and then the String database(https://string-db.org/cgi/input.pl)and Cytoscape3.5.1 software were used to construct the protein-protein interaction(PPI)network diagram of non-small cell lung cancer before and after drug resistance,so as to obtain the main DEGs.Kaplan-Meier survival analysis was performed on DEGs based on the survival software package on the R platform and the TCGA database(https://cancergenome.nih.gov/).DEGs related to NSCLC survival were selected,and molecular docking was further performed with the main structure of Astragalus polysaccharides.DEGs that can stably bind were selected as key genes and possible pathways were explored.Gefitinib-sensitive cell lines PC9 and HCC827 and gefitinib-stable acquired drugresistant cell lines PC9/GR and HCC827/GR were selected for in vitro verification.Each experiment intervened two groups of cells with the same intervention method.PC9 and HCC827 cells were selected and treated with different concentrations of gefitinib.The half maximal inhibitory concentration(IC50)of gefitinib on PC9 and HCC827 cells was detected by Cell Counting Kit-8(CCK-8)assay.The IC50 concentration of gefitinib calculated by each cell was applied to PC9/GR and HCC827/GR cells,and the authenticity of acquired gefitinib resistance in PC9/GR and HCC827/GR cells was detected by CCK-8 method.PC9/GR and HCC827/GR cells were treated with gefitinib IC50 concentration and different concentrations of APS,respectively.The IC50 concentration of APS was detected and calc ulated by CCK-8 method to determine the intervention concentration of experimental drugs.TLR4 knockdown cell lines PC9/GR+Si TLR4,HCC827/GR Si TLR4 and overexpression cell lines:PC9/GR+OE TLR4,HCC827/GR+OE TLR4 were constructed.The experiment was divided into two groups(PC9 and PC9/GR cells as an example).The first group was divided into four groups:PC9,PC9/GR,PC9/GR+Si TLR4,PC9/GR+OE TLR4;the second group was divided into four groups:PC9/GR,PC9/GR+OE TLR4,PC9/GR+APS+Gefitinib,PC9/GR+OE TLR4+APS+ Ge.Transwell assay and cell scratch assay were used to detect the invasion and migration ability of the second group of cells.Tunel assay was used to detect the apoptosis ability of the second group of cells.Real-time fluorescence quantitative method(qRt-PCR)was used to detect the mRNA expression levels of TLR4/MyD88 and EMT pathway related factors TLR4,MyD88,ECadherin,N-Cadherin and Vimentin in the second group of cells.Western blot(WB)was used to detect the expression levels of TLR4/MyD88 and EMT pathway-related proteins TLR4,MyD88,E-Cadherin,N-Cadherin and Vimentin-related factors in the first and second groups of cells.It was preliminarily verified that Astragalus polysaccharide combined with gefitinib interfered with EMT process through TLR4/MyD88 pathway and reversed non-small cell lung cancer.Results:1.A total of 290 DEGs were associated with acquired resistance to gefitinib in NSCLC,and the top ten DEGs with the highest correlation were CXCL8,TLR4,EGF,TLR2,SAA1,HP,CCR2,LYZ,S100A8,CSF1R.Among them,there are 5 genes related to the prognosis of NSCLC and can stably bind to APS,namely CXCL8,TLR4,TLR2,CCR2 and LYZ.2.CCK-8 assay showed that PC9 and HCC827 cells were sensitive to gefitinib,with IC50 values of 14.66 μmol/L and 20.56 μmol/L,respectively.After PC9/GR and HCC827/GR cells were treated with gefitinib(14.66 or 20.56 μmol/L),there was no significant difference in the survival rate of resistant strains.CCK-8 assay was used to detect cell viability after combined administration of gefitinib(14.66 or 20.56 μmol/L)and APS.It was found that 343.1 mg/L and 343.4 mg/L APS combined with gefitinib inhibited the proliferation of drug-resistant strains.The best effect,the difference was statistically significant.3.WB assay was used to detect the expression levels of TLR4,MyD88,E-Cadherin,NCadherin and Vimentin proteins in the first group of cells.It was found that compared with PC9(HCC827)cells,the high expression of TLR4/MyD88 pathway and EMT changes in PC9/GR(HCC827/GR)cells were increased,and the difference was statistically significant.With the knockdown and overexpression of TLR4,the changes of EMT were consistent with the changes of TLR4/MyD88 pathway,which proved that TLR4/MyD88 signaling pathway could regulate EMT process.4.Transwell assay,cell scratch assay and Tunel assay showed that the higher the expression of TLR4,the stronger the invasion,migration and anti-apoptosis of PC9/GR and HCC827/GR cells.Compared with PC9/GR(HCC827/GR)group,APS combined with gefitinib group could inhibit cell invasion,migration and induce apoptosis,and the difference was statistically significant.5.qRt-PCR and WB were used to detect the mRNA and protein expression levels of TLR4/MyD88 and EMT pathway-related molecules in the second group of cells.Compared with the PC9/GR(HCC827/GR)group,the APS combined with gefitinib group could inhibit the expression of TLR4,MyD88,N-Cadherin,Vimentin,and promote the expression of E-Cadherin.Conclusions:1.The TLR4/MyD88 signaling pathway is closely related to the acquired resistance of NSCLC to gefitinib,and the higher the expression of TLR4,the higher the malignancy of the cells.2.Astragalus polysaccharide combined with gefitinib can inhibit the invasion and migration of PC9/GR and HCC827/GR cells and induce apoptosis,which may be achieved by inhibiting the expression of TLR4 and regulating the TLR4/MyD88 signaling pathway to reg ulate the EMT process.3.Astragalus polysaccharide can reverse the acquired resistance of NSCLC to gefitinib.