非小细胞肺癌A549细胞基因组不稳定性和MYC基因突变在吉西他滨耐药中的作用  

Effects of genomic instability and MYC gene mutation of nonsmall cell lung cancer A549 cells on resistance of gemcitabine

作　　者：陈宗军 陈亚红 黄丽云 梁紫盈 CHEN Zongjun;CHEN Yahong;HUANG Liyun;LIANG Ziying(Department of Pharmacy,Second Affiliated Hospital,Hainan Medical College,Haikou 570311,China;Department of Respiratory,Second Affiliated Hospital,Hainan Medical College,Haikou 570311,China;Department of Pharmacy,First Affiliated Hospital,Hainan Medical College,Haikou 570216,China;Department of Physical Examination,Fourth People’s Hospital,Haikou City,Haikou 571100,China)

机构地区：[1]海南医学院第二附属医院药学部,海南海口570311 [2]海南医学院第二附属医院呼吸科,海南海口570311 [3]海南医学院第一附属医院药学部,海南海口570216 [4]海口市第四人民医院体检科,海南海口571100

出　　处：《吉林大学学报（医学版）》2024年第2期355-363,共9页Journal of Jilin University：Medicine Edition

基　　金：海南省卫健委2021年度海南省卫生健康行业科研项目(21A200045)。

摘　　要：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2mg·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),8mg·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 polⅡ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 polⅡandγH2AX 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 increase

关 键 词：吉西他滨 癌 非小细胞肺 基因组不稳定性 MYC扩增 MYC突变 

分 类 号：R734.2[医药卫生—肿瘤]