CMTM6对幽门螺杆菌感染的胃上皮细胞中PD-L1的作用

doi:10.19723/j.issn.1671-167X.2025.02.004

摘要/Abstract

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体(platelet activating factor receptor，PTAFR)的基因表达明显上调。结论: Hp感染上调胃黏膜细胞CMTM6水平，CMTM6发挥稳定PD-L1的作用；CMTM6基因敲除可能影响蛋白质泛素化降解、细胞表面受体表达。

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)

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)

中图分类号:

735.2

付玮, 宁静, 付伟伟, 张静, 丁士刚. CMTM6对幽门螺杆菌感染的胃上皮细胞中PD-L1的作用[J]. 北京大学学报（医学版）, 2025, 57(2): 245-252.

Wei FU, Jing NING, Weiwei FU, Jing ZHANG, Shigang DING. Effect of CMTM6 on PD-L1 in Helicobacter pylori infected gastric epithelial cells[J]. Journal of Peking University (Health Sciences), 2025, 57(2): 245-252.

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

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

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

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