靶向间充质干细胞微小RNA-26a和微小RNA-146a克服非小细胞肺癌靶向治疗耐药的研究

To overcome targeted therapy drug resistance in non-small cell lung cancer by targeting microRNA-26a and microRNA-146a from mesenchymal stem cells

目的探讨间充质干细胞(MSC)在非小细胞肺癌(NSCLC)抵抗表皮生长因子受体抑制剂(EGFR-TKI)耐药过程中的作用及其机制。方法HCC827及PC-9肺腺癌细胞与MSC共培养后给予吉非替尼处理,检测肺癌细胞凋亡变化;应用荧光定量聚合酶链反应(qPCR)检测共培养刺激后MSC细胞表达生长因子及细胞因子的情况;收集培养癌旁和远端正常肺组织来源MSC并行微小核糖核酸(miRNA)表达谱检测,并采用miRNA模拟物及抑制剂进行功能验证;此外,行聚合酶链反应微阵列分析(PCR Array)检测进一步探讨MSC调控NSCLC细胞抵抗吉非替尼的机制。两组数值间比较应用非配对t检验。结果肺癌来源MSC的miR-26a表达组低于正常肺组织来源MSC的miR-26a表达组(53.3%,t=4.626,P<0.01),而肺癌来源MSC的miR-146a表达组高于正常肺组织来源MSC的miR-146a表达组(1.83倍,t=4.895,P<0.01)。应用Transwell体系共培养肺癌细胞和MSC 48 h后发现,肺癌细胞(HCC827细胞)来源的miR-26a表达组明显低于MSC来源的miR-26a表达组(45.7%,t=1.866,P<0.05);且肺癌细胞(PC-9细胞)来源的miR-26a表达组也明显低于MSC来源的miR-26a表达组(62.0%,t=1.728,P<0.05),同时肺癌细胞(HCC827细胞)来源的肝细胞生长因子(HGF)表达组明显高于MSC来源的HGF表达组(3.88倍,t=2.014,P<0.05);且肺癌细胞(PC-9细胞)来源的HGF表达组也明显高于MSC来源的HGF表达组(5.29倍,t=2.197,P<0.05)。此外,MSC培养液可促进HCC827细胞可高表达转运蛋白和药物代谢基因三磷酸腺苷结合盒转运蛋白G2(ABCG2)(4.23倍)和细胞色素P450酶2C9(CYP2C9)(2.18倍)。结论肺癌来源的MSC通过异常表达miR-26a和miR-146a调控促肿瘤细胞因子增强NSCLC细胞对EGFR-TKI的抗药性。

Objective To explore the role and mechanism of mesenchymal stem cells(MSCs)in the resistance of non-small cell lung cancer against epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI).Methods EGFR mutated HCC827 and PC-9 lung adenocarcinoma cells were co-cultured with MSC and the apoptosis after EGFR-TKIgefitinib treatment was detected.Quantitative real-time polymerase chain reaction(qPCR)was used to examine the expression of growth factors and cytokines in MSCs after co-culture.MicroRNA expression profiles were performed for MSCfrom adjacent and distal normal lung tissues.MicroRNA mimics and inhibitors were used for functional verification.In addition,PCR Array detection was performed to further explore the mechanism of MSCs regulating the resistance of NSCLC to gefitinib.The GraphPad Prism Version 7.0 software was used for statistical analysis of data.The data are expressed as mean±standard deviation,and the unpaired t-test was used for the comparison between the two groups.Results The miR-26a expression in lung cancer-derived MSCs was lower than that in normal lung tissue-derived MSCs(53.3%,t=4.626,P<0.01),while the miR-146a expression in lung cancer-derived MSCs was higher than that in normal lung tissue-derived MSCs(1.83 times,t=4.895,P<0.01).After co-culture of lung cancer cells and MSCs with Transwell system for 48 h,it was found that the miR-26a expression derived from lung cancer cells(HCC827 cells)was significantly lower than the miR-26a expression derived from MSCs(45.7%,t=1.866,P<0.05).The miR-26a expression derived from lung cancer cells(PC-9 cells)was also significantly lower than that derived from MSCs(62.0%,t=1.728,P<0.05).The hepatocyte growth factor(HGF)expression derived from lung cancer cells(HCC827 cells)was significantly higher than that from the MSCs(3.88 folds,t=2.014,P<0.05).The lung cancer cells(PC-9 cells)-derived HGF expression was also significantly higher than MSCs-derived HGF expression(5.29 folds,t=2.197,P<0.05).HCC827 cells can highly express ATP-binding cassette transporter 2(ABCG2)(4.23 times)and cytochrome P450 proteins 2C9(CYP2C9)(2.18 times)after treatment with MSCs culture medium.Conclusion Lung cancer-derived MSCs expressed miR-26a and miR-146a abnormally,which could enhance the resistance of NSCLC cells to EGFR-TKI via tumor-promoting cytokines.