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.
Beijing Da Xue Xue Bao Yi Xue Ban. 2025 Apr 18;57(2):245-252. doi: 10.19723/j.issn.1671-167X.2025.02.004.
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 infection and the regulation of CMTM6 on PD-L1, and to analyze the mRNA expression differences before and after gene knock-out in infected gastric epithelial cells by microarray analysis.
The standard strain ATCC 26695 was co-cultured with human gastric epithelial cell GES-1 for 6, 24 and 48 hours, and the mRNA and protein levels of CMTM6 and PD-L1 were detected by real-time quantitative PCR and Western blot. Using CRISPR/Cas9 to construct gene knockout plasmid and knockout gene of GES-1 cells. was co-cultured with gene knockout and wild type GES-1 cells for 48 hours to detect PD-L1 transcription and protein level changes, and 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 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.
The mRNA and protein levels of CMTM6 and PD-L1 in GES-1 cells were significantly up-regulated after infection, and mRNA was most significantly up-regulated 48 hours after infection. After gene knockout, the gene transcription level of 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 gene knockout, 67 genes had more than two times of differential expression. The transcription levels of , , , , , and other genes were significantly down-regulated. The transcription levels of , , , , and other genes were significantly up-regulated. After gene knockout, ubiquitin-conjugating enzyme E2S (UBE2S) gene expression was significantly down-regulated, which might affect protein ubiquitination degradation. After gene knockout, adrenoceptor alpha 1B (ADRA1B), cholinergic receptor muscarinic 1 (M1), CHRM1, platelet activating factor receptor (PTAFR) gene expression was significantly up-regulated.
infection up-regulates the expression level of CMTM6 in gastric mucosa cells, and CMTM6 can stabilize PD-L1 and maintain the protein level of PD-L1. gene knockout may affect biological behaviors such as protein ubiquitination and cell surface receptor expression.
探讨感染后胃黏膜上皮细胞中趋化素样因子超家族成员6（CMTM6）和程序性死亡配体1（PD-L1）表达的变化以及CMTM6对PD-L1的调控作用，并通过基因芯片分析感染的胃上皮细胞基因敲除前后的mRNA表达差异。
将标准菌株ATCC 26695与人胃上皮细胞GES-1共培养6、24和48小时，采用实时定量PCR和蛋白质免疫印迹法检测CMTM6和PD-L1的mRNA和蛋白质水平。利用CRISPR/Cas9构建基因敲除质粒并敲除GES-1细胞的基因。将其与基因敲除的GES-1细胞和野生型GES-1细胞共培养48小时，检测PD-L1转录和蛋白质水平变化，并用蛋白酶体抑制剂MG-132处理基因敲除的GES-1细胞，检测PD-L1蛋白质水平变化。使用安捷伦人类ceRNA芯片2019检测与幽门螺杆菌（Hp）共培养48小时的基因敲除和野生型GES-1细胞中的差异表达基因，并通过京都基因与基因组百科全书（KEGG）数据库分析差异表达基因富集的信号通路。
感染后GES-1细胞中CMTM6和PD-L1的mRNA和蛋白质水平显著上调，感染后48小时mRNA上调最为显著。基因敲除后，感染的GES-1细胞的基因转录水平无明显变化，但PD-L1蛋白质水平显著下调，应用蛋白酶体抑制剂MG-132后PD-L1水平升高。基因敲除后，有67个基因表达差异超过两倍。、、、、、和等基因的转录水平显著下调。、、、和等基因的转录水平显著上调。基因敲除后，泛素结合酶E2S（UBE2S）基因表达显著下调，可能影响蛋白质泛素化降解。基因敲除后，肾上腺素能受体α1B（ADRA1B）、毒蕈碱型胆碱能受体1（M1）、CHRM1、血小板活化因子受体（PTAFR）基因表达显著上调。
感染上调胃黏膜细胞中CMTM6的表达水平，CMTM6可稳定PD-L1并维持其蛋白质水平。基因敲除可能影响蛋白质泛素化和细胞表面受体表达等生物学行为。
