| Background and ObjectiveLung cancer was one kind of malignant tumors with the highest incidence and mortality,which traditional treatment mainly included surgery,radiotherapy and chemotherapy.Non-small cell lung cancer(NSCLC),including lung adenocarcinoma,squamous cell carcinoma and so on,was 80-85%of the total lung cancer,and its new treatments determined by genetic background had received wide attention.Recently a large number of basic and clinical studies found:(1)Targeted therapy with tyrosine kinase inhibitors(TKIs)in NSCLC patients with EGFR gene mutations had significantly prolonged progression-free survival(PFS)and had a significant reduction in side effects than traditional chemotherapy.(2)Although targeted therapy,using of gefitinib,erlotinib and other TKIs,had gradually become one of the standard treatment regimens for advanced NSCLC with EGFR mutations,but most of NSCLC patients with EGFR-TKIs targeted therapy would inevitably have targeted drug resistance in about 12 months,which greatly affected the use of EGFR-TKIs in the treatment of NSCLC.(3)Although the EGFR gene T790M secondary mutation,HER2 and MET amplification,BRAF and KRAS mutations had been identified as the causes of EGFR-TKIs resistance in NSCLC,but those gene changes were not detected in a significant number of NSCLC patients with EGFR-TKIs resistance.Therefore,further study of about EGFR-TKIs resistance markers and mechanism had a very important theoretical significance and potential application value for reversion of EGFR-TKIs resistance and improving the therapeutic effect in NSCLC.In this study,we first continuously induced HCC827 cell line by gefitinib to establish the EGFR-TKIs drug-resistant cell mode,and then the differential gene expression profiles and the difference spectrum of miRNAs related with EGFR-TKIs resistance were established by high-throughput gene chip and next generation sequencing.Next the role of molecular targets obtained by screening was confirmed in clinical samples,in vivo and in vitro,and the drug-related signal regulation network was identified.Finally,the mechanism of EGFR-TKIs resistance in non-small cell lung cancer was elucidated by means of bioinformatics analysis,IP experiment,Chip experiment and double luciferase reporter gene experiment.The study hopes to find new EGFR-TKIs resistance related molecular targets that can help for development of EGFR-TKIs drug resistance diagnostic kit and reversal of EGFR-TKIs resistance.Part 1 The establishment of EGFR-TKIs resistance cell model in NSCLC and screening of drug resistance related moleculesMethodsTo establish the EGFR-TKIs drug-resistant cell mode named HCC827/AR by inducing HCC827 cell line using gefitinib,and the EGFR-TKIs resistance of HCC827/AR was confirmed by drug sensitivity test and subcutaneous tumorigenesis test in mice;Phenotypic characteristics of drug resistance cell model were detected by plate cloning experiment,flow cytometry and so on;EGFR,KRAS,BRAF,ALK,ROS1,HER2,MET gene mutation or amplification status were compared between parental cell HCC827 and drug-resistant cell HCC827/AR;Gene expression profiles of HCC827 and HCC827/AR cells were detected by gene microarray and verified by Q-PCR,and resistance-related signaling pathways were simulated through bioinformatics analysis;To knockdown the expression of key signal molecule PFTK1(CDK14)in HCC827/AR cells and detect the sensitivity change of the cells to gefitinib;Next generation sequencing was used to detect differential expression of exosomes miRNAs in HCC827 and HCC827/AR and results were validated by Q-PCR;A number of miRNAs,which were highly expressed in HCC827/AR cells,were selected as candidate drug-resistant miRNAs.Then the gefitinib resistance of HCC827 or HCC827/AR was detected after translocating mimics of the candidate drug-resistant miRNA into HCC827 or translocating corresponding miRNA inhibitor into HCC827/AR for determing EGFR-TKIs resistance-related miRNAs;Results1.Establishment of EGFR-TKIs resistance cell model HCC827/AR1.1 EGFR-TKIs drug-resistant cell line HCC827/AR was induced successfully.Compared with HCC827,the IC50 values of the HCC827/AR to gefitinib,erlotinib and alfatinib were increased from 1.00±0.04μM,0.59±0.01μM and 5.43±0.17μM to 28.54±2.31μM,47.36±0.58μM,29.53±0.64μM(all P<0.05),respectively.In vivo experiments also confirmed that HCC827 and HCC827/AR cells were sensitive and resistant to gefitinib.1.2 The EGFR-TKIs sensitivity-related gene status of HCC827 and HCC827/AR were similar(Such as EGFR gene exon 19 deletion mutations existed in two cell lines;EGFR gene exons 18,20and 21 were wild type in two cell lines;No mutations,fusions and amplifications were found in KRAS,BRAF,ALK,ROS1,HER2 and MET genes in two cell lines.),which suggested that some unknown genes were involved in the tolerance of EGFR-TKIs in HCC827/AR cells.2.Establishment of the differential gene expression profiles related with EGFR-TKIs resistance and screening of candidate drug-related target genes2.1 Chips assay results showed that 1083 genes were up-regulated more than 2-fold and 1036genes were down-regulated(less than 0.5-fold)in drug-resistant cell HCC827/AR compared with that of parental cell HCC827.The results of Q-PCR quantitative analysis of 5 up-regulated genes and 5 down-regulated genes were in accordance with the results of the chip,suggesting that the established differential expression profiles of resistance-related genes are of reference value.2.2 Drug-resistant molecular analog signaling pathway was obtained through bioinformatics analysis of these differentially expressed genes and PFTK1 gene,whose expression was up-regulated by 19 times in HCC827/AR cells,was the hinge molecule in the analog signaling pathway.The sensitivity to gefitinib in HCC827/AR cells knocked down PFTK1 increased by3.03±0.33 folds(P=0.02).2.3 The NGS results of exosomes miRNAs in HCC827 and HCC827/AR cell culture medium showed that 130 miRNAs were up-regulated and 8 miRNAs were down-regulated in drug-resistant cell HCC827/AR compared with that of parental cell HCC827.And the expression of miRNA200c-3p in HCC827/AR cells increased by 7.49±0.53 folds(P=3.23E-5).The sensitivity to gefitinib in HCC827/AR cells knocked down miRNA200c-3p increased by 2.27±0.03 folds(P=5.87E-5),suggesting that miRNA200c-3p was involved in the regulation of drug sensitivity.Part 2 The role of PFTK1 in the resistance of EGFR-TKIs in NSCLCMethodsThe relationship between PFTK1 expression and prognosis in 1926 lung cancer patients was analyzed by using Kaplan Meier plotter database;The expression of PFTK1 in lung cancer tissues before and after EGFR-TKIs resistance was detected by immunohistochemistry;Q-PCR and WB were used to detect the expression of PFTK1 and its related signaling pathway in HCC827 and HCC827/AR cells;Knockdown the expression of PFTK1 in HCC827/AR cells,detect the change of gefitinib sensitivity,key genes expression and the phenotype in HCC827/AR cells of before and after PFTK1 knockdown;The change of gefitinib sensitivity was detected after overexpression of PFTK1 in HCC827 cells;Knockdown the expression ofβ-catenin in HCC827/AR cells,detect the change of gefitinib sensitivity in cells of before and afterβ-catenin knockdown;Knockdown the expression of MET in HCC827/AR cells,detect the change of gefitinib sensitivity,key genes expression and the phenotype in HCC827/AR cells of before and after MET knockdown;After HCC827/AR cells were treated with MK2206,a specific AKT inhibitor,the change of gefitinib sensitivity,key genes expression and the phenotype of the cells were examined before and after treatment.Q-PCR and WB were used to screen the lung cancer cell lines with high expression of PFTK1.Then the expression of PFTK1 and its related signal pathway were detected in the cells with high expression of PFTK1 whose PFTK1 was interfered,in the same time the resistance to gefitinib also was detected;The change of gefitinib resistance of HCC827/AR after interference with PFTK1 was also detected by subcutaneous tumorigenesis test in mice.Results1.Relationship between PFTK1 expression and prognosis or drug tolerance in lung cancer1.1 The PFTK1 scores tested by immunohistochemistry assay were 8.02±1.27 and 8.85±2.89,respectively,in lung cancer tissues before and after EGFR-TKIs resistance.And the expression of PFTK1 in lung cancer tissues after EGFR-TKIs resistance was significantly higher than that in cancer tissues before EGFR-TKIs resistance(P=0.04).1.2 The median survival time of PFTK1 high expression group was significantly shorter than that of PFTK1 low expression group(P=0.02),suggesting that lung cancer patients with PFTK1-overexpressing had shorter survival and poorer prognosis.2.Role of PFTK1 in EGFR-TKIs drug resistance in vitro2.1 HCC827/AR cell line with stable PFTK1 interference was successfully constructed,whose relative IC50 to gefitinib decreased to 0.38±0.03(P=7.57E-6),whose m RNA expression of MET,AKT 1,AKT3 and PFTK1 were significantly decreased(all P<0.05),whose MET,P-MET,AKT,P-AKT,PFTK1 andβ-catenin protein expression were decreased,whose cell proliferation was decreased but apoptosis was increased.When PFTK1 was overexpressed in HCC827 cells,the relative inhibitory rate of gefitinib to the cells was reduced to 0.71±0.08(P=4.18E-3)compared with the control group,suggesting an increase in drug resistance.The m RNA expression of MET,AKT3 and PFTK1 in HCC827/AR cells were significantly higher than that in HCC827 cells(all P<0.05);MET,P-MET,AKT,P-AKT,PFTK1,β-catenin protein expression in HCC827/AR cells were higher than that in HCC827 cells.2.2 Whenβ-catenin was interfered in HCC827/AR cells,the relative inhibitory rate of gefitinib to cells was increased to 1.37±0.02(P=5.89E-5)compared with the control group,suggesting a decrease in drug resistance.2.3 HCC827/AR cell line with stable MET interference was successfully constructed,whose relative IC50 to gefitinib decreased to 0.44±0.05(P=5.44E-5),whose m RNA expression of MET,AKT 1,AKT3 and PFTK1 were significantly decreased(all P<0.05),whose MET,P-MET,AKT,P-AKT,PFTK1 andβ-catenin protein expression were decreased,whose cell proliferation was decreased but apoptosis was increased.2.4 Compared with the control group,the relative IC50 of HCC827/AR cells to gefitinib decreased to 0.10±1.56E-3(P=1.42E-5)when the AKT inhibitor MK2206 was used in combination with gefitinib.Q-PCR results showed that the m RNA expression of MET,AKT1,AKT3 and PFTK1 in MK2206-treated HCC827/AR cells was significantly decreased(all P<0.05).The results of WB showed that the protein expression of P-MET,P-AKT,PFTK1 andβ-catenin in MK2206-treated HCC827/AR cells,whose cell proliferation tested by flow cytometry was increased,was decreased.2.5 The m RNA and protein of PFTK1 were highly expressed in lung cancer H1650 and A549/DDP cells.After blocking PFTK1 expression in H1650 and A549/DDP cells,the relative inhibitory rate of gefitinib to cells was increased to2.73±0.06(P=2.45E-5)and 2.18±0.22(P=1.13E-3),respectively,suggesting a decrease in drug resistance;And the m RNA expression of MET,AKT 1,AKT3 and PFTK1 were significantly decreased(all P<0.05),and the protein expression of MET and PFTK1 was decreased.3.Role of PFTK1 in EGFR-TKIs drug resistance in vivoIn experiment of the mice subcutaneous tumorigenesis,the mean tumor volume of the HCC827/AR-PFTK1 sh RNA group mice treated with gefitinib gavage simultaneously was 286.32±121.41 mm~3,which was significantly smaller than that of HCC827/AR-Control group mice(851.30±236.30 mm~3,P=0.02)and HCC827/AR-PFTK1 sh RNA group mice(631.64±103.07mm~3,P=0.02),suggesting that HCC827/AR cells whose PFTK1 expression was interfered also reduced the resistance to gefitinib in vivo.Part 3 Mechanism of PFTK1-mediated resistance to EGFR-TKIs in NSCLCMethodsBioinformatics predicted the binding site of TCF/LEF family transcription factors in the promoter region of the MET gene;Chip experiments confirmed whether the predicted promoter binded to the promoter region of the MET gene;Double luciferase reporter assay confirmed the effect of the corresponding promoter on MET gene expression;To confirm the change of drug resistance of HCC827/AR to gefitinib after interfering with MET by in vivo tumorigenesis in mice;The expression of MET in lung cancer was detected by immunohistochemistry and the survival analysis of MET was done in NSCLC patients.The correlation analysis between PFTK1 expression and MET expression was done in NSCLC.The survival analysis of PFTK1 combined with MET was done in NSCLC patients;IP experiment combined with phosphorylated antibody test to determine whether PFTK1 and AKT protein binding and whether the phosphorylation level of PFTK1was increased;Bioinformatics predicted transcription factors that could bind to the PFTK1promoter region;The expression of FOXO3a in HCC827 and HCC827/AR cells were detected by Q-PCR and WB;Chip experiments demonstrated whether FOXO3a could bind to the promoter region of the PFTK1 gene;The effect of FOXO3a on PFTK1 gene expression was confirmed by double luciferase reporter assay;Bioinformatics simultaneously analyzed the binding site of TCF/LEF family transcription factors in the miRNA200c-3p coding gene promoter region and the target genes regulated by miRNA200c-3p;Q-PCR and WB were used to detect the expression of TP53 in HCC827 and HCC827/AR cells;Q-PCR was used to detect the expression of miRNA200c-3p in HCC827/AR cells after interference PFTK1;The expression of TP53 in HCC827/AR cells after interference PFTK1 was detected by Q-PCR and WB;Q-PCR was used to detect the expression of TP53 in HCC827/AR cells after interferingβ-catenin;Q-PCR and WB were used to detect the expression of TP53 in HCC827 transfected with miRNA200c-3p mimics and HCC827/AR transfected with miRNA200c-3p inhibitors,and then cell’s changes in apoptosis were detected by flow cytometry;Chip experiments demonstrated whether the predicted promoter could bind to the promoter region of miRNA200c-3p coding gene;The effect of the corresponding promoter on the expression of miRNA200c-3p gene was confirmed by double luciferase reporter assay;The effect of miRNA200c-3p on TP53 gene expression was also confirmed by double luciferase reporter assay.Results1.Mechanism of PFTK1 modulating MET byβ-catenin/TCF3 Pathway to mediate EGFR-TKIs drug resistance1.1 Bioinformatics predicted the presence of TCF3 transcription factor binding sites in the promoter region of the MET gene.PCR amplification with primer 4 was successful in Chip experiments usingβ-catenin antibody to precipitate the chromatin in HCC827/AR cells;Results of double luciferase reporter gene experiment showed that overexpression of TCF3 could increase the relative luciferase activity in the experimental group to 2.88±0.15(P=3.34E-5),suggesting that TCF3 could promote the expression of MET gene.It was known that PFTK1 could activate Wnt/β-catenin signaling pathway.Therefore,the above results showed that PFTK1 could promote the expression of MET gene by activating Wnt pathway to increase freeβ-catenin which could transfer into the nuclear and combine with TCF3.1.2 In experiment of the mice subcutaneous tumorigenesis,the mean tumor volume of the HCC827/AR-MET sh RNA group mice treated with gefitinib gavage simultaneously was245.52±124.37 mm~3,which was significantly smaller than that of HCC827/AR-Control group mice(851.30±236.30 mm~3,P=0.02)and HCC827/AR-MET sh RNA group mice(644.15±128.18mm~3,P=0.02),suggesting that HCC827/AR cells whose MET expression was interfered also reduced the resistance to gefitinib in vivo.1.3 The MET scores tested by immunohistochemistry assay were 2.98±3.55 and 6.23±3.68,respectively,in lung cancer tissues before and after EGFR-TKIs resistance.The expression of MET in lung cancer tissues after EGFR-TKIs resistance were significantly higher than that in lung cancer tissues before EGFR-TKIs resistance(P=0.02).The correlation(positive correlation)between PFTK1 and MET was gradually increased in lung cancer tissues before and after EGFR-TKIs resistance.The median survival time of MET high expression group was significantly shorter than that of MET low expression group(P<0.05).And the median survival time of lung cancer patients with high-expression of PFTK1 and MET was significantly shorter than that of lung cancer patients with low-expression of PFTK1 and MET(P<0.05).2.Mechanism of PFTK1 interacted with AKT to mediate EGFR-TKIs drug resistance2.1 The results of IP experiments showed that PFTK1 and AKT proteins could be detected in the proteins captured by PFTK1 antibody or AKT antibody.The phosphorylated PFTK1 in HCC827/AR cells was increased by pan phosphorylated antibody test.The results indicated that AKT bound to PFTK1 and promoted its phosphorylation activation.2.2 Bioinformatics predicted the presence of FOXO3a transcription factor binding sites in the promoter region of the PFTK1 gene;The expression of FOXO3a m RNA in HCC827/AR cells was higher than that in HCC827 cells(P<0.05),but the FOXO3a protein in HCC827/AR cells was lower than that in HCC827 cells;PCR amplification with some of primers was successful in Chip experiments using FOXO3a antibody to precipitate the chromatin in HCC827 and HCC827/AR cells;Results of double luciferase reporter gene experiment showed that overexpression of FOXO3a could increase the relative luciferase activity in the experimental group to 2.47±0.13(P=2.13E-4),suggesting that FOXO3a could promote the expression of PFTK1 gene.It was known that the activation of PI3K/AKT signaling pathway could promote the phosphorylation of FOXO3a that promoted the degradation of FOXO3a.Therefore,the above results indicated that the phosphorylation of AKT could negative feedback regulate PFTK1 expression through lowering FOXO3a by promoting FOXO3a phosphorylation.3.Mechanism of miR200c-3p regulated TP53 to mediate EGFR-TKIs drug resistance3.1 Bioinformatics predicted the presence of TCF3 transcription factor binding sites in the promoter region of the miRNA200c-3p coding gene and TP53 was one of miRNA200c-3p downstream target genes;Both Q-PCR and WB results showed that TP53 expression in HCC827/AR cells was lower than that in HCC827 cells;In HCC827/AR cells with interference PFTK1,the expression of miRNA200c-3p was decreased(P<0.05),and the TP53 m RNA was increased(P<0.05),bur the TP53 protein did not change significantly;The TP53 m RNA was increased significantly(P<0.05)in HCC827/AR cells with interferenceβ-catenin;The m RNA and protein of TP53 in HCC827 cells transfected with miRNA200c-3p mimics was decreased and apoptosis was reduced also;The m RNA and protein of TP53 in HCC827/AR cells transfected with miRNA200c-3p inhibitors was increased and apoptosis was increased also;3.2 PCR amplification with primer 1 was successful in Chip experiments usingβ-catenin antibody to precipitate the chromatin in HCC827/AR cells;Results of double luciferase reporter gene experiment showed that overexpression of TCF3 could increase the relative luciferase activity in the experimental group to1.66±0.22(P=0.02),suggesting that TCF3 could promote the expression of miRNA200c-3p gene.Results of another double luciferase reporter gene experiment showed that transfection of miRNA200c-3p mimics could decrease the relative luciferase activity in the experimental group to 0.6±0.06(P=2.57E-3),suggesting that miRNA200c-3p could inhibit the expression of TP53 gene.Above results suggested that PFTK1 could activate the wnt pathway to increase the expression of miRNA200c-3p by increasing the amount of free beta-catenin,which could transfer into the nucleus and combine with TCF3,whereas the highly expressed miRNA200c-3p could bind to the TP53 3’UTR region to inhibit the expression and translation of the TP53 gene that could inhibit apoptosis and participate in PFTK1-mediated EGFR-TKIs resistance.Conclusion1.PFTK1 mediated EGFR-TKIs targeted therapy resistance in HCC827/AR cell line of NSCLC.2.PFTK1 mediated EGFR-TKIs resistance by transcriptional activation of MET viaβ-catenin.3.MET/PI3K/AKT promoted PFTK1 phosphorylation.4.MET/PI3K/AKT/FOXO3a inhibited PFTK1 expression at the transcriptional level.5.PFTK1 mediated EGFR-TKIs resistance by transcriptional activation of miR200c-3p viaβ-catenin.6.miR200c-3p mediated EGFR-TKIs resistance by targeted inhibition of TP53... |
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