目的 探讨非小细胞肺癌（NSCLC）A549细胞基因不稳定性和MYC基因突变在吉西他滨耐药中的作用，并阐明其作用机制。 方法 采用2 mg·L^－1吉西他滨持续作用A549细胞（A549组），构建耐药细胞株A549R（A549R组），并将si-NC和si-MYC转染至A549R细胞，作为si-NC A549R组和si-MYC A549R组。采用CCK-8法检测不同浓度（0、1、2、4、8、16和32 mg·L^－1）吉西他滨对各组细胞的抑制率，流式细胞术检测各组细胞凋亡率。转录组测序技术（RNA-seq）和京都基因与基因组百科全书（KEGG）信号通路富集分析A549组及A549R组细胞中的差异表达基因，实时荧光定量PCR（RT-qPCR）法检测A549组和A549R组细胞中基因组不稳定性相关基因错配修复基因2（MSH2）、错配修复基因6（MSH6）和DNA修复蛋白RAD50（RAD50）的表达水平，Western blotting法检测基因组不稳定性相关蛋白MYC原癌基因（MYC）和磷酸化组蛋白（γH2AX）的表达水平，染色质免疫共沉淀（ChIP）法检测RNA polⅡ和γH2AX在MYC基因上的富集程度，PCR法扩增并检测A549R细胞MYC基因突变情况。 结果 与A549组比较，2、4、8、16和32 mg·L^－1吉西他滨作用后A549R组细胞抑制率降低（P<0.05），8 mg·L^－1吉西他滨作用后细胞凋亡率降低（P<0.05）。与A549组比较，A549R组细胞中有234个mRNAs表达水平升高，205个mRNAs表达水平降低，其中错配修复相关基因（MSH2和MSH6）、RAD50和MYC表达水平明显升高（P<0.05）。KEGG信号通路富集分析，表达水平升高基因主要参与非同源末端连接、mRNA监测途径和DNA复制等信号通路。与A549组比较，A549R组细胞中MYC和γH2AX蛋白表达水平升高（P<0.05）。ChIP法检测，RNA pol Ⅱ和γH2AX在MYC转录起始位点及exon 2上富集程度增加，MYC exon 2上存在G254A基因突变。与si-NC A549R组比较，si-MYC A549R组细胞中MYC mRNA和蛋白表达水平降低（P<0.05），2、4、8、16和32 mg·L^－1吉西他滨作用后si-MYC A549R组细胞抑制率升高（P<0.05），细胞凋亡率增加（P<0.05）。 结论 对吉西他滨耐药的NSCLC细胞中A549R基因组不稳定性增加，MYC基因发生突变和扩增，而敲减MYC可以恢复A549R细胞对吉西他滨的敏感性。

Objective To discuss the effects of genomic instability and MYC gene mutation of non-small cell lung cancer （NSCLC） A549 cells on the resistance of gemcitabine， and to clarify the mechanism. Methods The A549 cells were continuously treated with 2 mg·L^－1 of gemcitabine （A549 group） to establish the resistant cell line A549R （A549R group）， and si-NC and si-MYC were transfected into the A549R cells to regarded as si-NC A549R group and si-MYC A549R group， respectively. CCK-8 assay was used to detect the inhibitory rates of the cells in various groups after treated with various concentrations of gemcitabine （0， 1， 2， 4， 8， 16， and 32 mg·L^－1）；flow cytometry was used to detect the apoptotic rates of the cells in various groups； transcriptome sequencing technology （RNA-seq） and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were used to identify the differentially expressed genes in the A549 and A549R cells； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of muts homology 2 （MSH2）， muts homology 6 （MSH6）， and recombinant DNA repair protein RAD50（RAD50） in the A549 and A549R cells； Western blotting method was used to detect the expression levels of genome instability-related proteins MYC proto-oncogene （MYC） and phosphorylated H2AX （γH2AX） in the cells in various groups； chromatin immunoprecipitation （ChIP） was used to detect the enrichment of RNA pol Ⅱ and γH2AX on the MYC gene； PCR method was used to amplify and detect the mutations in MYC gene in the A549R cells. Results Compared with A549 group， the inhibitory rates of the A549R cells in A549R group treated with 2， 4， 8， 16， and 32 mg·L^－1 gemcitabine were decreased， and the apoptotic rate of the cells after treated with 8 mg·L^－1 gemcitabine was decreased （P<0.05）. Compared with A549 group， a total of 234 mRNAs in the cells in A549R group were upregulated and 205 mRNAs in the cells were downregulated， and the expression levels of mismatch repair-related genes （MSH2 and MSH6）， RAD50， and MYC were significantly increased （P<0.05）. The KEGG signaling pathway enrichment analysis showed that the upregulated genes were mainly involved in non-homologous end-joining， mRNA surveillance pathway， and DNA replication signaling pathways. Compared with A549 group， the expression levels of MYC and γH2AX proteins in the cells in A549R group were increased （P<0.05）.The ChIP assay results showed that the enrichment of RNA pol Ⅱ and γH2AX at the MYC transcription start site and exon 2， with a G254A mutation identified in MYC exon 2 was increased. Compared with si-NC A549R group， the expression levels of MYC mRNA and protein in the cells in si-MYC A549R group were decreased （P<0.05）， the inhibitory rates of the cells after treated with 2， 4， 8， 16， and 32 mg·L^－1 gemcitabine were increased（P<0.05）， and the apoptotic rates were increased （P<0.05）. Conclusion The genome instability of the A549R NSCLC cells resistant to gemcitabine is increased， and the mutations and amplification occur in the MYC gene. Knockdown of MYC can restore the sensitivity of A549R cells to gemcitabine.