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目的建立对克唑替尼耐药的非小细胞肺癌细胞株,并对其耐药性进行鉴定,初步筛选耐药相关的miRNAs。方法采用克唑替尼药物浓度递增的方法建立人耐药细胞株NCI-H3122CR,并通过细胞生长曲线、药物敏感性、细胞凋亡和细胞周期的检测评价NCI-H3122CR细胞株的耐药性。第二代基因测序法和RT-PCR检测耐药后microRNA表达量的变化。结果成功建立了克唑替尼耐药细胞株H3122CR,耐药指数为9.86。流式细胞仪检测结果显示H3122CR细胞凋亡率比H3122细胞低,并呈现浓度依赖性的趋势。耐药细胞株的细胞周期分布产生了显著的变化。当前的第二代测序和RT-PCR检测结果显示:miR-30a-5p、miR-374c-5p和miR-125b-5p表达上调,miR-31-5p、miR-10a-5p和miR-200c-3p表达下调。结论构建的H3122CR耐药细胞模型对克唑替尼有一定的耐药性,并且耐药后部分miRNAs表达量产生显著的变化。
Objective To establish a cell line of non-small cell lung cancer resistant to crizotinib, and to identify its drug resistance, and to screen for drug resistance-related miRNAs. Methods NCI-H3122CR cells were established by increasing concentration of crizotinib. The drug resistance of NCI-H3122CR cells was evaluated by cell growth curve, drug sensitivity, apoptosis and cell cycle. The second generation of gene sequencing and RT-PCR detection of resistant microRNA expression changes. Results The crizotinib-resistant cell line H3122CR was successfully established and the drug resistance index was 9.86. Flow cytometry results showed that the apoptosis rate of H3122CR cells was lower than that of H3122 cells in a concentration-dependent manner. Cell cycle distribution of drug-resistant cell lines produced significant changes. The current second-generation sequencing and RT-PCR results showed that miR-30a-5p, miR-374c-5p and miR-125b-5p were up- 3p expression down regulation. Conclusion The constructed H3122CR resistant cell model has certain resistance to crizotinib, and the expression of some miRNAs in drug-resistant cells has a significant change.