CMTM6对幽门螺杆菌感染的胃上皮细胞中PD-L1的作用  

Effect of CMTM6 on PD-L1 in Helicobacter pylori infected gastric epithelial cells

作　　者：付玮 宁静 付伟伟 张静[1] 丁士刚[1] FU Wei;NING Jing;FU Weiwei;ZHANG Jing;DING Shigang(Department of Gastroenterology,Peking University Third Hospital/Beijing Key Laboratory for Helicobacter Pylori Infection and Upper Gastrointestinal Diseases,Beijing 100191,China)

机构地区：[1]北京大学第三医院消化科,幽门螺杆菌感染及上胃肠疾病防治研究北京市重点实验室,北京100191

出　　处：《北京大学学报(医学版)》2025年第2期245-252,共8页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81870386)。

摘　　要：目的:探索幽门螺杆菌(Helicobacter pylori,Hp)感染后胃黏膜上皮细胞中含MARVEL结构域的CKLF样因子6(CKLF-like MARVEL transmembrane domain-containing 6,CMTM6)、程序性死亡配体(programmed death-ligand 1,PD-L1)表达水平变化及CMTM6对PD-L1的调控作用,并通过微列阵分析探索CMTM6基因敲除前后Hp感染的胃黏膜上皮细胞mRNA表达差异变化情况。方法:将Hp标准菌株ATCC 26695与人胃黏膜上皮细胞GES-1共培养6 h、24 h及48 h,通过实时荧光定量PCR及免疫印迹法检测CMTM6及PD-L1表达水平。利用CRISPR/Cas9技术构建CMTM6基因敲除质粒并敲除GES-1细胞的CMTM6基因。将Hp分别与CMTM6基因敲除和野生型GES-1细胞共培养48 h,检测PD-L1转录及蛋白质水平的变化,并利用蛋白酶体抑制剂MG-132处理CMTM6基因敲除GES-1细胞,检测PD-L1蛋白水平变化。利用Agilent Human ceRNA Microarray 2019对与Hp共培养48 h的CMTM6基因敲除和野生型GES-1细胞进行mRNA微列阵分析,得到差异表达基因并通过日本京都基因与基因组百科(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库分析差异表达基因富集的信号通路。结果:Hp感染后,GES-1细胞的CMTM6、PD-L1的mRNA及蛋白水平均明显上调,CMTM6 mRNA在感染后48 h上调最明显。CMTM6基因敲除后,Hp感染的GES-1细胞CD274基因转录水平无明显变化,但PD-L1蛋白水平明显下调,应用蛋白酶体抑制剂MG-132处理后PD-L1水平回升。CMTM6基因敲除后,67个基因表达差异达到2倍以上,其中TMEM68、FERMT3、GPR142、ATP6V1FNB、NOV、UBE2S等基因转录水平明显下调,PCDHGA6、CAMKMT、PDIA2、NTRK3、SPOCK1等基因转录水平明显上调。CMTM6基因敲除后,编码泛素结合酶E2S(ubiquitin-conjugating enzyme E2S,UBE2S)的基因表达明显下调,可能影响蛋白质泛素化降解。CMTM6基因敲除后,编码肾上腺素能受体α1B(adrenoceptor alpha 1B,ADRA1B)、乙酰胆碱毒蕈碱受体M1(cholinergic receptor muscarinic 1,CHRM1)及血小板活化因子受Objective:To explore the changes of CKLF-like MARVEL transmembrane domain-containing 6(CMTM6)and programmed death-ligand 1(PD-L1)expression in gastric mucosal epithelial cells after Helicobacter pylori infection and the regulation of CMTM6 on PD-L1,and to analyze the mRNA expression differences before and after CMTM6 gene knock-out in helicobacter pylori infected gastric epithelial cells by microarray analysis.Methods:The standard Helicobacter pylori strain ATCC 26695 was co-cultured with human gastric epithelial cell GES-1 for 6,24 and 48 hours,and the mRNA and protein le-vels of CMTM6 and PD-L1 were detected by real-time quantitative PCR and Western blot.Using CRISPR/Cas9 to construct CMTM6 gene knockout plasmid and knockout CMTM6 gene of GES-1 cells.Helicobacter pylori was co-cultured with CMTM6 gene knockout and wild type GES-1 cells for 48 hours to detect PD-L1 transcription and protein level changes,and CMTM6 gene knockout GES-1 cells were treated with the proteasome inhibitor MG-132 to detect the changes in PD-L1 protein levels.Agilent Human ceRNA Microarray 2019 was used to detect the differentially expressed genes in CMTM6 gene knockout and wild-type GES-1 cells co-cultured with Hp for 48 hours,and the signal pathway of differentially expressed genes enrichment was analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG)database.Results:The mRNA and protein levels of CMTM6 and PD-L1 in GES-1 cells were significantly up-regulated after Helicobacter pylori infection,and CMTM6 mRNA was most significantly up-regulated 48 hours after infection.After CMTM6 gene knockout,the CD274 gene transcription level of Helicobacter pylori infected GES-1 cells did not change significantly,but PD-L1 protein level was significantly down-regulated,and the PD-L1 level increased after the application of proteasome inhibitor MG-132.After CMTM6 gene knockout,67 genes had more than two times of differential expression.The transcription levels of TMEM68,FERMT3,GPR142,ATP6V1FNB,NOV,UBE2S and other genes were significantly down-regu

关 键 词：胃黏膜 上皮细胞 幽门螺杆菌 含MARVEL域蛋白质类(CMTM6) B7-H1抗原(PD-L1) 

分 类 号：R735.2[医药卫生—肿瘤]