[Effect of CMTM6 on PD-L1 in infected gastric epithelial cells].

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 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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.