收稿日期: 2021-11-16
网络出版日期: 2025-04-12
基金资助
国家自然科学基金(81870386)
版权
Effect of CMTM6 on PD-L1 in Helicobacter pylori infected gastric epithelial cells
Received date: 2021-11-16
Online published: 2025-04-12
Supported by
the National Natural Science Foundation of China(81870386)
Copyright
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)
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 . DOI: 10.19723/j.issn.1671-167X.2025.02.004
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.
| 1 | Oster P , Vaillant L , Riva E , et al. Helicobacter pylori infection has a detrimental impact on the efficacy of cancer immunotherapies[J]. Gut, 2022, 71 (3): 457- 466. |
| 2 | Correa P . Human gastric carcinogenesis: A multistep and multifactorial process. First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention[J]. Cancer Res, 1992, 52 (24): 6735- 6740. |
| 3 | Asaka M , Sugiyama T , Nobuta A , et al. Atrophic gastritis and intestinal metaplasia in Japan: Results of a large multicenter study[J]. Helicobacter, 2001, 6 (4): 294- 299. |
| 4 | Zhao M , Liu Q , Liu W , et al. MicroRNA140 suppresses Helicobacter pyloripositive gastric cancer growth by enhancing the antitumor immune response[J]. Mol Med Rep, 2019, 20 (3): 2484- 2492. |
| 5 | Xie G , Li W , Li R , et al. Helicobacter pylori promote B7-H1 expression by suppressing miR-152 and miR-200b in gastric cancer cells[J]. PLoS One, 2017, 12 (1): e0168822. |
| 6 | Holokai L , Chakrabarti J , Broda T , et al. Increased programmed death-ligand 1 is an early epithelial cell response to Helicobacter pylori infection[J]. PLoS Pathog, 2019, 15 (1): e1007468. |
| 7 | Beswick EJ , Pinchuk IV , Das S , et al. Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells[J]. Infect Immun, 2007, 75 (9): 4334- 4341. |
| 8 | Wu YY , Lin CW , Cheng KS , et al. Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection[J]. Clin Exp Immunol, 2010, 161 (3): 551- 559. |
| 9 | Burr ML , Sparbier CE , Chan YC , et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity[J]. Nature, 2017, 549 (7670): 101- 105. |
| 10 | Mezzadra R , Sun C , Jae LT , et al. Identification of CMTM6 and CMTM4 as PD-L1 protein regulators[J]. Nature, 2017, 549 (7670): 106- 110. |
| 11 | Guan X , Zhang C , Zhao J , et al. CMTM6 overexpression is associated with molecular and clinical characteristics of malignancy and predicts poor prognosis in gliomas[J]. EBioMedicine, 2018, 35, 233- 243. |
| 12 | Tian Y , Sun X , Cheng G , et al. The association of CMTM6 expression with prognosis and PD-L1 expression in triple-negative breast cancer[J]. Ann Transl Med, 2021, 9 (2): 131. |
| 13 | Ishihara S , Iwasaki T , Kohashi K , et al. The association between the expression of PD-L1 and CMTM6 in undifferentiated pleomorphic sarcoma[J]. J Cancer Res Clin Oncol, 2021, 147 (7): 2003- 2011. |
| 14 | Li X , Chen L , Gu C , et al. CMTM6 significantly relates to PD-L1 and predicts the prognosis of gastric cancer patients[J]. PeerJ, 2020 (8): e9536. |
| 15 | Wang Z , Peng Z , Liu Q , et al. Co-expression with membrane CMTM6/4 on tumor epithelium enhances the prediction value of PD-L1 on anti-PD-1/L1 therapeutic efficacy in gastric adenocarcinoma[J]. Cancers (Basel), 2021, 13 (20): 5175. |
| 16 | Chen L , Yang QC , Li YC , et al. Targeting CMTM6 suppresses stem cell-like properties and enhances antitumor immunity in head and neck squamous cell carcinoma[J]. Cancer Immunol Res, 2020, 8 (2): 179- 191. |
| 17 | Huang X , Xiang L , Wang B , et al. CMTM6 promotes migration, invasion, and EMT by interacting with and stabilizing vimentin in hepatocellular carcinoma cells[J]. J Transl Med, 2021, 19 (1): 120. |
| 18 | Jin MH , Nam AR , Park JE , et al. Therapeutic co-targeting of WEE1 and ATM downregulates PD-L1 expression in pancreatic cancer[J]. Cancer Res Treat, 2020, 52 (1): 149- 166. |
| 19 | Tanaka E , Miyakawa Y , Kishikawa T , et al. Expression of circular RNA CDR1AS in colon cancer cells increases cell surface PDL1 protein levels[J]. Oncol Rep, 2019, 42 (4): 1459- 1466. |
| 20 | Yamamoto Y , Kakizaki M , Shimizu T , et al. PD-L1 is induced on the hepatocyte surface via CKLF-like MARVEL transmembrane domain-containing protein 6 up-regulation by the anti-HBV drug entecavir[J]. Int Immunol, 2020, 32 (8): 519- 531. |
| 21 | Ho JY , Lu HY , Cheng HH , et al. UBE2S activates NF-κB signaling by binding with IκBα and promotes metastasis of lung adenocarcinoma cells[J]. Cell Oncol (Dordr), 2021, 44 (6): 1325- 1338. |
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