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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (2): 245-252. doi: 10.19723/j.issn.1671-167X.2025.02.004

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

Wei FU, Jing NING, Weiwei FU, Jing ZHANG*(), Shigang DING*()   

  1. Department of Gastroenterology, Peking University Third Hospital; Beijing Key Laboratory for Helicobacter Pylori Infection and Upper Gastrointestinal Diseases, Beijing 100191, China
  • Received:2021-11-16 Online:2025-04-18 Published:2025-04-12
  • Contact: Jing ZHANG, Shigang DING E-mail:sihuizhang@sina.com;dingshigang222@163.com
  • Supported by:
    the National Natural Science Foundation of China(81870386)

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Abstract:

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 levels 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-regulated. The transcription levels of PCDHGA6, CAMKMT, PDIA2, NTRK3, SPOCK1 and other genes were significantly up-regulated. After CMTM6 gene knockout, ubiquitin-conjugating enzyme E2S (UBE2S) gene expression was significantly down-regulated, which might affect protein ubiquitination degradation. After CMTM6 gene knockout, adrenoceptor alpha 1B (ADRA1B), cholinergic receptor muscarinic 1 (M1), CHRM1, platelet activating factor receptor (PTAFR) gene expression was significantly up-regulated. Conclusion: Helicobacter pylori 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. CMTM6 gene knockout may affect biological behaviors such as protein ubiquitination and cell surface receptor expression.

Key words: Gastric mucosa, Epithelial cells, Helicobacter pylori, MARVEL domain-containing proteins (CMTM6), B7-H1 antigen (PD-L1)

CLC Number: 

  • 735.2

Figure 1

The mRNA and protein expressions of CMTM6 and PD-L1 in GES-1 cells infected with Hp 26695 detected by RT-qPCR and Western blot * *P < 0.01, vs. 0 h. Hp, Helicobacter pylori; PD-L1, programmed death-ligand 1; CMTM6, CKLF-like MARVEL transmembrane domain-containing 6."

Table 1

sgRNA sequence targeting CMTM6 gene"

sgRNA Sequence (5′→ 3′)
sgRNA-F CACCGCGGCCCGAGGCGATGGAGAA
sgRNA-R AAACTTCTCCATCGCCTCGGGCCGC

Figure 2

Verification of CMTM6 gene knockout in GES-1 cells A, sgRNA-F fragment in CMTM6 gene knockout plasmid detected by Sanger sequencing; B, CMTM6 mRNA and CMTM6 protein expressions in CMTM6 gene wild-type/knockout GES-1 cells detected by RT-qPCR and Western blot. * *P < 0.01. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; WT, wild-type; KO, knockout."

Figure 3

mRNA and protein expressions of CMTM6, PD-L1 in CMTM6 gene wild-type/knockout GES-1 cells before and after Hp 26695 infection detected by RT-qPCR and Western blot * *P < 0.01. CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; Hp, Helicobacter pylori; WT, wild-type; KO, knockout; PD-L1, programmed death-ligand 1."

Figure 4

The level of PD-L1 protein in GES-1 cells after CMTM6 gene knockout and protease inhibitor MG-132 treatment were detected by Western blot CMTM6, CKLF-like MARVEL transmembrane domain-containing 6; KO, knockout; PD-L1, programmed death-ligand 1."

Figure 5

Cluster analysis and volcanic map of CMTM6 gene knockout/wild-type GES-1 differentially expressed genes Each point in the graph represents a differentially expressed gene, with the horizontal axis representing the logarithmic value (log2 FC) of the fold diffe-rence in gene expression between two sets of samples, and the vertical axis representing the statistically significant negative logarithmic value [-lg (P value)] of gene expression changes. The larger the absolute value of the horizontal axis, the greater the difference in expression levels between the two groups; The larger the absolute value of the vertical axis, the more significant the differential expression."

Table 2

Up-regulated genes after CMTM6 gene knockout"

Rank Gene symbol Fold change P
1 PCDHGA6 3.962 0.009
2 CAMKMT 3.478 0.007
3 PDIA2 3.430 0.001
4 NTRK3 3.371 0.001
5 SPOCK1 3.085 0.001

Table 3

Down-regulated genes after CMTM6 gene knockout"

Rank Gene symbol Fold change P
1 TMEM68 -3.782 0.005
2 FERMT3 -3.269 0.012
3 GPR142 -3.059 0.003
4 ATP6V1FNB -2.938 0.002
5 NOV -2.809 0.003

Figure 6

KEGG enrichment analysis of differentially expressed genes PPAR, peroxisome proliferator-activated receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes."

Table 4

KEGG enrichment analysis gene list"

Rank Term description Gene symbols P
1 Neuroactive ligand-receptor interaction ADRA1B, CHRM1, PTAFR, CGA 0.019 3
2 Calcium signaling pathway ADRA1B, CHRM1, PTAFR 0.023 9
3 Melanogenesis GNAO1, WNT10A 0.046 9
4 Cholinergic synapse GNAO1, CHRM1 0.056 4
5 Serotonergic synapse SLC6A4, GNAO1 0.057 3
6 Other types of O-glycan biosynthesis B3GLCT 0.073 3
7 Protein export IMMP2L 0.076 5
8 Retrograde endocannabinoid signaling NDUFB1, GNAO1 0.084 3
9 Adrenergic signaling in cardiomyocytes ADRA1B, CACNG8 0.087 4
10 Phototransduction SAG 0.092 4
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