MiR-206 Involved In Gefitinib Acquired Drug-resistance In Non-small Cell Lung Cancer Patients Through Modulating IL-6 Expression

Lung cancer is the major cause for cancer-related death worldwide with a five-year survival of less than 20%.In China mainland,lung cancer related mortality and morbidity ranks as the first position.As the major histological type of lung cancer,non-small cell lung cancer(NSCLC)accounts for about 80-85%of these patients.Unfortunately,most of the cases are at the advanced stage upon diagnosis,losing the opportunity for radical surgery.For these patients,chemotherapy is considered the major treatment option.Nowadays,epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)have been considered as effective target therapy agents for treating advanced NSCLC.Compared with conventional chemotherapy,EGFR-TKIs could significnatly delay the disease progression and improve the quality of life.EGFR-TKIs alternate conventional treatment options and have been listed as the first-line medicine.However,there are really acquired drug tolerances in the first,second and third generation of agents within one year after administration,which hampers its clinical application and the survival benefits of the patients.Thus,more and more studies have focused on illustrating the drug resistance mechanisms of EGFR-TKIs,together with screening new treatment targets.In this study,we screened the differentially expressed miRNA in NSCLC patients through bioinformatics.Our data showed that miR-206 was significantly down-regulated in lung cancer tissues compared with adjacent tissues.Signaling pathway analysis showed that the target genes of miR-206 were extensively associated with the interleukin(IL)related signaling pathway.After a comprehensive literature search,we found that IL-6 and miR-206 could affect the acquired drug tolerance in EGFR-TKIs.To date,there are no studies on illustrating the roles of miR-206 in IL-6 regulation in the EGFR-TKI acquired resistance in NSCLC patients.We hypothesize that miR-206 may directly act on IL-6,which then modulated the IL-6 mediated EGFR-TKIs acquired resistance.1.ObjectiveIn this study,we aimed to detect the expression of miR-206 and IL-6 and their correlation in EGFR-mutated NSCLC tissues,in order to illustrate the relationship between their expression and acquired resistance of Gefitinib.On this basis,we could identify the potential mechanism of how miR-206 mediated the Gefitinib resistance in lung cancer cells with EGFR mutations in the presence of IL-6,which paves the way for the identification of new targets for overcoming the Gefitinib resistance.2.Methods2.1 Lung cancer database analysis based on bioinformaticsWe downloaded the raw chip data of GSE36681 from GEO database(Gene expression omnibus,GEO;http://www.ncbi.nlm.nih.gov/geo/),and analyzed the differentially expressed genes based on GEO2R differentially expressed software.The miRTarBase(http://mirtarbase.mbc.nctu.edu.tw/)based on data validation was used for predicting target genes of differentially expressed miRNA.Then KEGG(https://david.ncifcrf.gov/)and Reactome(https://reactome.org/)are used for the enrichment analysis for the predicted miRNA target genes.2.2 Expression and correlation of miR-206 and IL-6 in tissue and serum samplesCancer tissue samples and serum samples from 37 lung cancer patients served as the experimental group,and samples obtained from tissues that were 2 cm from 14 patients with pulmonary chondromal hamartoma were utilized as control.Quantitative real time polymerase chain reaction(RT-qPCR)was.utilized to determine the expression of miR-206 and IL-6 in tissue samples.Enzyme linked immunosorbent assay(ELISA)was used to determine serum IL-6 expression.2.3 Roles of gefitinib resistance in lung cancer cells presenting EGFR mutationsPC-9 and HCC827 cell lines were pretreated using IL-6,and then were subject to drug sensitivity tests based on CCK-8 analysis.Then IL-6 pretreated PC-9 and HCC827 cell lines were subject to Western blot analysis for expression of phosphorylated protein in Jak1/Stat3 signaling pathway.Finally,RT-qPCR was performed to determine the expression of miR-206 in these cells.2.4 Effects of miR-206 on Gefitinib resistance in IL-6 pretreated EGFR mutation cellsIL-6 pretreated cells were transiently transfected with miR-206 mimics,and then CCK-8 assay was conducted to determine cellular viability.Flow cytometry was carried out to determine the cellular death.The cell colony capacity was determined.Western blot analysis was conducted to determine expression of phosphorylated protein in Jakl/Stat3 signaling pathway.Finally,IL-6 pretreated cells that were transiently transfected with miR-206 mimics were treated using Stat3 agonist(colivelin),followed by determination of cellular viability using CCK-8 assay.2.5 Regulatory effects of miR-206 on IL-6RNA-Chip test was used to enrich Ago2/RJSC complex,and then RT-qPCR was conducted to determine mRNA abundance.Endogenous IL-6 expression in miR-206 transfected lung cancer cells was determined based on Western blot analysis.The effects of miR-206 on IL-6 were validated using luciferase reporting gene.2.6 Regulatory effects of IL-6 on miR-206RT-qPCR was utilized to determine expression of miR-206,pre-miR-206 and pri-miR-206 in IL-6 treated PC-9 cells.Co-immunoprecipitation was carried out using p-Stat3 specific antibody,and Drosha and GGCR8 expression was determined using Western blot analysis.In IL-treated PC-9 cells adding Jak kinase inhibitor(pyridine 6,P6),expression of miR-206,pre-miR-206 and pri-miR-206 was determined using Western blot analysis,p-Stat3 and RT-qPCR,respectively.In IL-6 treated PC-9 cells,RNA-Chip was performed after adding p-Stat3 and Drosha antibody.Then Western blot analysis was conducted to determine expression of p-Stat3 and Drosha protein.In the presence of co-precipitation of p-Stat3 and Drosha antibody,RT-qPCR was used to determine pri-miR-206 expression.2.7 In tumor-bearing mice,we validated that miR-206 inhibited PC-9 oncogenicity and increased Gefitinib sensitivity.IL-6 pretreated PC-9 cells that were transfected using miR-206 or NC mimics were injected into scapula BALB/c nude mice to induce tumor.Upon tumor volume reaching 50 mm3,Gefitinib was utilized,followed by animal scarification on day 4,together with tumor proliferation and expression of IL-6/Jakl/Stat3.2.8 Statistical analysisAll data were presented in the form of mean ± standard deviation.The difference between groups was analyzed by Two-tailed T test.P<0.05 was considered to be Statistically significant.Spearman correlation analysis was performed to analyze the correlation between miR-206 expression and serum IL-6.All the data were analyzed using Graphpad Prism 7.0 software.3.Results3.1 MiR-206 was significantly down-regulated in the GSE36681 datasets,which extensively involved in IL-related signaling pathway through regulating expression of target genes.Differentially expressed analysis showed that there were 204 miRNAs that were significantly expressed in NSCLC tissues,including 112 that were up-regulated and 92 that were down-regulated.A total of 122 target genes of miR-206 were identified from miRTarBase.KEGG analysis showed that miR-206 involved in regulating PI3K-Akt,AMPK and Jak-Stat signaling pathways through modulating the expression of target genes.Reactome analysis showed that miR-206 extensively involved in IL-related signaling pathway.3.2 There was significant down-regulation of miR-206 and up-regulation of IL-6 in NSCLC tissues;miR-206 was negatively correlated with IL-6.In NSCLC tissues presenting EGFR mutations,miR-206 expression was significantly lower compared to that of normal control(P<0.01).In NSCLC patients compare with control group the expression of plasma IL-6 was remarkable high(P<0.01),miR-206 was negatively correlated with IL-6(r=-0.7762,P<0.001).In 14 NSCLC cancer tissues,compare with the adjacent cancer tissues the expression of IL-6 was significantly high.3.3 IL-6 induced Gefitinib resistance in lung cancer cells presenting EGFR mutationBoth PC-9 and HCC827 cells were sensitive to Gefitinib under normal culture conditions,however,.in the presence of IL-6,both cells presented tolerance,with the IC50 of 2.16±0.26 μM and 1.17±0.19 μM,respectively.Upon IL-6 treatment,there was significant elevation in the pJak1(P<0.05)and pStat3(P<0.01).Compared with normal cultured cells,there was significant down-regulation of miR-206 in IL-6 treated cells.In contrast,upon treating with Gefitinib,there was further decline of miR-206 expression(P<0.05).3.4 MiR-206 inhibited Gefitinib tolerance of EGFR mutation cells through modulating IL-6/Jak1/Stat3 axis.After treating with IL-6 in cells transfected using miR-206 mimics,the cells showed recovery in sensitivity of Gefitinib,with decline of IC50 of 0.16±0.08 μM and 0.19± 0.11 μM,respectively.Gefitinib could induce significant elevation in the proportion of apoptotic cells,which was about 2-fold compared to that of normal control.The cell colony showed significant decline,which indicated that Gefitinib could significantly inhibit the cell colony.MiR-206 mimics could obviously inhibit the expression of p-Stat3 and p-Jak1 rather than the Stat3 and Jak1 total protein(P<0.05).Upon treating with colivelin,the cellular viability showed decline,and showed Gefitinib resistance in partial cells.3.5 MiR-206 could regulate IL-6 in a target manner.In this section,we determined the relative extent of mRNA of 15 predicted genes in the RNA-Chip test.The enrichment of IL-6 in miR-206 over-expressed cells was significantly higher than that of the normal control(P<0.01).In PC-9 and HCC827 cells transfected using miR-206 mimics,the expression of IL-6 showed significant down-regulation.Luciferase reporting system indicated that cells with co-transfection of WT plasmid and miR-206 mimics showed significant decline(44.7%)in the luciferase activity compared with the control group subject to co-transfection of WT plasmid and NC mimics(P<0.01).The luciferase activity in cells transfected using WT plasmid and miR-206 inhibitor showed significant increase(26.1%)compared with those co-transfected with WT plasmid and NC inhibitor(P<0.01).In cells transfected using WT plasmid,miR-206 mimics co-transfection induced significant decline in luciferase activity,while there was no significant elevation in the luciferase activity after transfection with miR-206 inhibitor.These indicated that IL-6 gene was the target gene of miR-206.3.6 IL-6 could regulate miR-206 in a feedback manner.In PC-9 cells treated using IL-6,there was significant down-regulation of pre-miR-206 and miR-206,while there was no significant changes in pri-miR-206.After IL-6 treatment,there was no significant up-regulation of Drosha and DGCR8 in PC-9 cells.Western blot analysis showed that there was no interaction between p-Stat3 and Drosha/DGCR8.The pyridone 6 could significantly decline the expression of p-Stat3,and inhibit IL-6/Jak1/Stat3 signaling pathway,together with significant down-regulation of pri-miR-206 and up-regulation of miR-206 and pre-miR-206.In RNA-Chip test,there was high expression of pri-miR-206 in Stat3 antibody precipitation,while its content showed significant decline in Drosha antibody precipitation(P<0.01).3.7 MiR-206 could inhibit the cancer cell growth and improve the Gefitinib sensitivity.Compared with normal control group,there was significant decline in tumor growth in miR-206 group and miR-206+Gef group(P<0.05),meanwhile,there was significant elevation in IL-6(P<0.01).In addition,there was significant decline in the phosphorylation of Jakl and Stat3(P<0.01).4.ConclusionIn NSCLC,there was significant down-regulation of miR-206 and up-regulation of IL-6.There was a negative correlation between them.IL-6 could induce Gefitinib tolerance in EGFR mutation lung cancer cells through modulating Jak1/Stat3 signaling pathway.MiR-206 could directly target IL-6,which then reversed the Gefitinib tolerance in EGFR mutation lung cancer cells.IL-6 could down-regulate the miR-206 expression by inhibiting Stat3 phosphorylation which interacts with pri-miR-206 to inhibit the processing and maturation.