miR-19a通过抑制c-MET的表达逆转HCC827细胞吉非替尼耐药
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摘要
目的明确miR-19a在调控HCC827细胞吉非替尼耐药中的作用及机制,探索miR-19a作为非小细胞肺癌(NSCLC)患者吉非替尼耐药新靶点的理论依据。方法收集非小细胞肺癌患者15例在吉非替尼耐药前后的外周血,通过Real-time PCR检测其中miR-19a的表达;在吉非替尼敏感的HCC827细胞中降低miR-19a表达,并加入吉非替尼,通过CCK8法检测其对细胞活力的影响;在构建成功的HCC827吉非替尼耐药细胞株(HCC827GR)中过表达miR-19a,并加入吉非替尼,通过CCK8法检测其对细胞活力的影响;在HCC827及HCC827GR细胞中分别降低或过表达miR-19a,通过Western blot检测c-MET蛋白表达水平;在HCC827细胞中同时降低miR-19a和c-MET表达,并加入不同浓度吉非替尼,通过CCK8法检测各组细胞活力。结果在吉非替尼耐药的NSCLC患者外周血中,miR-19a呈明显低表达状态;在HCC827细胞中降低miR-19a表达,吉非替尼对细胞活力的抑制效果明显下降(P<0.01);在HCC827GR细胞中过表达miR-19a,吉非替尼对细胞活力的抑制效果明显增强(P<0.01);在HCC827细胞中降低miR-19a表达,能明显促进c-MET蛋白的表达;相反,在HCC827GR细胞中过表达miR-19a,则明显抑制c-MET蛋白的表达;在HCC827细胞中同时降低miR-19a和c-MET表达,并加入不同浓度的吉非替尼,低表达c-MET能够抵消miR-19a对细胞活力的影响。结论 miR-19a在非小细胞肺癌细胞HCC827吉非替尼耐药中发挥着重要作用,其机制可能是通过抑制c-MET蛋白水平的表达。
Objective To clarify the role of miR-19 a in gefitinib resistance and its molecular biological mechanisms in HCC827 cells.To explore the theoretical basis of miR-19 a as a new target for reversal of gefitinib resistance in NSCLC patients.Methods Before and after gefitinib resistance,miR-19 a expression was detected in peripheral blood by real-time PCR.Then cell viability was measured by CCK8 assay after cells were transfected with miR-19 a inhibitor and treated with different doses of gefitinib in HCC827 cells.On the other hand,cell viability was measured by CCK8 assay after cells were transfected with miR-19 a mimics and treated with different doses of gefitinibin in HCC827 GR cells.Furthermore,c-MET protein level was detected in HCC827 or HCC827 GR cells after the cells were transfected with miR-19 a inhibitor or mimics.Finally,HCC827 cells were transfected with miR-19 a inhibitor and si-c-MET,cell viability was measured by CCK8 after cells were treated with different doses of gefitinib.Results miR-19 a expression was significantly down-regulated in peripheral blood of patients with gefitinib resistance compared with gefitinib-sensitive patients.Inhibition of gefitinib on cell viability was significantly decreased after HCC827 cells were transfected with miR-19 a inhibitor(P<0.01).However,inhibition of gefitinib on cell viability was significantly enhanced after HCC827 GR cells were transfected with miR-19 a mimics(P<0.01).Down-regulation of miR-19 a raised c-MET protein level in HCC827 cells while over-expression of miR-19 a reduced c-MET protein level in HCC827 GR cells.Low expression of c-MET could counteract the effect of miR-19 a on cell proliferation after HCC827 cells were treated with different doses of gefitinib.Conclusion miR-19 a plays an important role in gefitinib-resistance of HCC827 cells,which might be related with inhibition of c-MET protein expression.
Objective To clarify the role of miR-19 a in gefitinib resistance and its molecular biological mechanisms in HCC827 cells.To explore the theoretical basis of miR-19 a as a new target for reversal of gefitinib resistance in NSCLC patients.Methods Before and after gefitinib resistance,miR-19 a expression was detected in peripheral blood by real-time PCR.Then cell viability was measured by CCK8 assay after cells were transfected with miR-19 a inhibitor and treated with different doses of gefitinib in HCC827 cells.On the other hand,cell viability was measured by CCK8 assay after cells were transfected with miR-19 a mimics and treated with different doses of gefitinibin in HCC827 GR cells.Furthermore,c-MET protein level was detected in HCC827 or HCC827 GR cells after the cells were transfected with miR-19 a inhibitor or mimics.Finally,HCC827 cells were transfected with miR-19 a inhibitor and si-c-MET,cell viability was measured by CCK8 after cells were treated with different doses of gefitinib.Results miR-19 a expression was significantly down-regulated in peripheral blood of patients with gefitinib resistance compared with gefitinib-sensitive patients.Inhibition of gefitinib on cell viability was significantly decreased after HCC827 cells were transfected with miR-19 a inhibitor(P<0.01).However,inhibition of gefitinib on cell viability was significantly enhanced after HCC827 GR cells were transfected with miR-19 a mimics(P<0.01).Down-regulation of miR-19 a raised c-MET protein level in HCC827 cells while over-expression of miR-19 a reduced c-MET protein level in HCC827 GR cells.Low expression of c-MET could counteract the effect of miR-19 a on cell proliferation after HCC827 cells were treated with different doses of gefitinib.Conclusion miR-19 a plays an important role in gefitinib-resistance of HCC827 cells,which might be related with inhibition of c-MET protein expression.
引文
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[14] Sun G,Lu Y,Li Y,et al.miR-19a protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via PTEN/PI3K/p-Akt pathway[J].Biosci Rep,2017,37(6):pii:BSR20170899.
[15] Li J,Yang S,Yan W,et al.MicroRNA-19 triggers epithelial-mesenchymal transition of lung cancer cells accompanied by growth inhibition[J].Lab Invest,2015,95(9):1056-1070.
[2] Tartarone A,Lazzari C,Lerose R,et al.Mechanisms of resistance to EGFR tyrosine kinase inhibitors gefitinib/erlotinib and to ALK inhibitor crizotinib[J].Lung Cancer,2013,81(3):328-336.
[3] Balak M N,Gong Y,Riely G J,et al.Novel D761Y and commonsecondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors[J].Clin Cancer Res,2006,12(21):6494-6501.
[4] Yoshida T,Zhang G,Smith M A,et al.Tyrosine phosphoproteomics identifies both codrivers andcotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lungcancer[J].Clin Cancer Res,2014,20(15):4059-4074.
[5] Engelman J A,Zejnullahu K,Mitsudomi T,et al.MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling[J].Science,2007,316(5827):1039-1043.
[6] Gadgeel S M,Wozniak A.Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistantnon-small-cell lung cancer[J].Clin Lung Cancer,2013,14(4):322-332.
[7] Cortot A B,Repellin C E,Shimamura T,et al.Resistance to irreversible EGF receptor tyrosine kinase inhibitors through a multistep mechanism involving the IGF1R pathway[J].Cancer Res,2013,73(2):834-843.
[8] Lin Y,Wang X,Jin H.EGFR-TKI resistance in NSCLC patients:mechanisms and strategies[J].Am J Cancer Res,2014,4(5):411-435.
[9] Sun K,Lai E C.Adult-specific functions of animal microRNAs[J].Nat Rev Genet,2013,14(8):535-548.
[10] Kong Y W,Ferland-McCollough D,Jackson T J,et al.microRNAs in cancer management[J].Lancet Oncol,2012,13(6):e249-e258.
[11] Lin Q,Chen T,Lin Q,et al.Serum miR-19a expression correlates with worse prognosis of patients with non-small cell lungcancer[J].J Surg Oncol,2013,107(7),767-771.
[12] Cao X,Lai S,Hu F,et al.miR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met[J].Sci Rep,2017,7(1):2939.
[13] Wu K,Chang Q,Lu Y,et al.Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells[J].Oncotarget,2013,4(12):2430-2438.
[14] Sun G,Lu Y,Li Y,et al.miR-19a protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via PTEN/PI3K/p-Akt pathway[J].Biosci Rep,2017,37(6):pii:BSR20170899.
[15] Li J,Yang S,Yan W,et al.MicroRNA-19 triggers epithelial-mesenchymal transition of lung cancer cells accompanied by growth inhibition[J].Lab Invest,2015,95(9):1056-1070.