基于YTHDF2介导凋亡相关因子降解途径研究牙龈卟啉单胞菌协助食管癌免疫逃逸

doi:10.12122/j.issn.1673-4254.2024.06.17

摘要/Abstract

摘要：

目的探讨牙龈卟啉单胞菌（Pg）感染食管癌细胞后对YTHDF2及凋亡相关因子（Fas）的影响，阐明其促进免疫逃逸可能的调控机制。方法运用免疫组织化学法及Western blot法检测Pg感染食管癌与YTHDF2及Fas表达变化；运用免疫共沉淀验证YTHDF2和Fas蛋白间的相互作用；将体外培养的Pg感染后的KYSE150细胞通过慢病毒转染分为对照组（转染si-NC）和实验组（转染si-YTHDF2），Western blotting检测两组细胞中YTHDF2、组织蛋白酶B（CTSB）及Fas、FasL蛋白的表达水平；对Pg感染的KYSE150细胞用组织蛋白酶抑制剂（E64）进行处理，Western blotting分别检测抑制剂处理前后YTHDF2、CTSB、Fas及FasL蛋白的表达变化情况；Pg感染前后及E64处理的KYSE150细胞与人外周血单个核细胞（PBMC）共培养，运用流式细胞术检测T细胞相关效应分子的表达情况。结果免疫组化法及Western blotting结果显示，Pg感染后的组织或细胞中YTHDF2的表达增强，而Fas表达减弱（P<0.001）；免疫共沉淀结果显示，YTHDF2和Fas之间可直接相互作用；Western blotting结果显示，si-YTHDF2组细胞中CTSB表达量减低，而Fas及FasL的表达量高于si-NC组（P<0.001）；用E64处理细胞后，CTSB表达减弱，YTHDF2表达无影响，而Fas及FasL的表达增强；流式细胞术结果显示，与PBMC共培养的细胞中，GranzymeB及Ki67表达由强到弱分别为Pg阴性、Pg阳性+E64、Pg阳性，而PD-1表达情况相反（P<0.001）。结论 Pg感染依赖YTHDF2调节Fas的表达，协助食管癌免疫逃逸，从而促进食管癌发生发展，表明YTHDF2可能是一个新的调控食管癌免疫逃逸的关键分子。

关键词: 食管鳞状细胞癌, 牙龈卟啉单胞菌, m⁶A甲基化阅读蛋白YTHDF2, 凋亡相关因子, 肿瘤免疫

Abstract:

Objective To investigate the effect of Porphyromonas gingivalis (Pg) infection on immune escape of oesophageal cancer cells and the role of YTHDF2 and Fas in this regulatory mechanism. Methods We examined YTHDF2 and Fas protein expressions in esophageal squamous cell carcinoma (ESCC) tissues with and without Pg infection using immunohistochemistry and in Pg-infected KYSE150 cells using Western blotting. The interaction between YTHDF2 and Fas was investigated by co-immunoprecipitation (Co-IP). Pg-infected KYSE150 cells with lentivirus-mediated YTHDF2 knockdown were examined for changes in expression levels of YTHDF2, cathepsin B (CTSB), Fas and FasL proteins, and the effect of E64 (a cathepsin inhibitor) on these proteins were observed. After Pg infection and E64 treatment, KYSE150 cells were co-cultured with human peripheral blood mononuclear cells (PBMCs), and the expressions of T cell-related effector molecules were detected by flow cytometry. Results ESCC tissues and cells with Pg infection showed significantly increased YTHDF2 expression and lowered Fas expression. The results of Co-IP demonstrated a direct interaction between YTHDF2 and Fas. In Pg-infected KYSE150 cells with YTHDF2 knockdown, the expression of CTSB was significantly reduced while Fas and FasL expressions were significantly increased. E64 treatment of KYSE150 cells significantly decreased the expression of CTSB without affecting YTHDF2 expression and obviously increased Fas and FasL expressions. Flow cytometry showed that in Pg-infected KYSE150 cells co-cultured with PBMCs, the expressions of Granzyme B and Ki67 were significantly decreased while PD-1 expression was significantly enhanced. Conclusion Pg infection YTHDF2-dependently regulates the expression of Fas to facilitate immune escape of esophageal cancer and thus promoting cancer progression, suggesting the key role of YTHDF2 in regulating immune escape of esophageal cancer.

Key words: esophageal squamous cell carcinoma, Porphyromonas gingivalis, m⁶A methylated reading protein YTHDF2, Fas, tumor immunity

杨泽, 张秀森, 张旭东, 柳颖, 张嘉诚, 原翔. 基于YTHDF2介导凋亡相关因子降解途径研究牙龈卟啉单胞菌协助食管癌免疫逃逸[J]. 南方医科大学学报, 2024, 44(6): 1159-1165.

Ze YANG, Xiusen ZHANG, Xudong ZHANG, Ying LIU, Jiacheng ZHANG, Xiang YUAN. Porphyromonas gingivalis infection facilitates immune escape of esophageal cancer by enhancing YTHDF2-mediated Fas degradation[J]. Journal of Southern Medical University, 2024, 44(6): 1159-1165.

图/表 3

图2 YTHDF2可与Fas相互作用

Fig.2 Co-immunoprecipitation for detecting interaction between YTHDF2 and Fas proteins.

图3 YTHDF2介导Fas蛋白通过组织蛋白酶途径降解

Fig.3 YTHDF2 mediates Fas protein degradation through the tissue protease pathway. A, B: Western blotting for detecting changes in YTHDF2, CTSB, Fas, and FasL protein expressions in KYSE150 cells after lentivirus transfection. C, D: Western blotting for detecting changes in protein expression of CTSB, YTHDF2, Fas, and FasL in KYSE150 cells after treatment with the tissue protease inhibitor (E64). ***P<0.001.

图4 Pg依赖YTHDF2-Fas途径抑制食管癌中T细胞的免疫效应

Fig. 4 Immunomodulatory effect of Pg infection mediated by the YTHDF2-Fas pathway on T cells in esophageal cancer. A-C: Flow cytometric analysis of the effects of Pg infection on expressions of Granzyme B, Ki-67, and PD-1 in T cells co-cultured with KYSE150 cells. D-F: Statistical charts of flow cytometry results. ***P<0.001.

参考文献 36

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49.
2	Pennathur A, Gibson MK, Jobe BA, et al. Oesophageal carcinoma[J]. Lancet, 2013, 381(9864): 400-12.
3	Zhou JC, Sun KX, Wang SM, et al. Associations between cancer family history and esophageal cancer and precancerous lesions in high-risk areas of China[J]. Chin Med J, 2022, 135(7): 813-9.
4	Xie SH, Lagergren J. Risk factors for oesophageal cancer[J]. Best Pract Res Clin Gastroenterol, 2018, 36/37: 3-8.
5	Cancer Genome Atlas Research Network, Analysis Working Group: Asan University, BC Cancer Agency, et al. Integrated genomic characterization of oesophageal carcinoma[J]. Nature, 2017, 541(7636): 169-75.
6	Deng JY, Chen H, Zhou DZ, et al. Comparative genomic analysis of esophageal squamous cell carcinoma between Asian and Caucasian patient populations[J]. Nat Commun, 2017, 8(1): 1533.
7	Lamont RJ, Fitzsimonds ZR, Wang HZ, et al. Role of Porphyromonas gingivalis in oral and orodigestive squamous cell carcinoma[J]. Periodontol 2000, 2022, 89(1): 154-65.
8	Gao SG, Yang JQ, Ma ZK, et al. Preoperative serum immunoglobulin G and A antibodies to Porphyromonas gingivalis are potential serum biomarkers for the diagnosis and prognosis of esophageal squamous cell carcinoma[J]. BMC Cancer, 2018, 18(1): 17.
9	Yuan X, Liu YW, Li GF, et al. Blockade of immune-checkpoint B7-H4 and lysine demethylase 5B in esophageal squamous cell carcinoma confers protective immunity against P. gingivalis infection[J]. Cancer Immunol Res, 2019, 7(9): 1440-56.
10	Zhuang HZ, Yu B, Tao D, et al. The role of m6A methylation in therapy resistance in cancer[J]. Mol Cancer, 2023, 22(1): 91.
11	Lan Q, Liu PY, Haase J, et al. The critical role of RNA m6A methylation in cancer[J]. Cancer Res, 2019, 79(7): 1285-92.
12	Zaccara S, Jaffrey SR. A Unified Model for the Function of YTHDF Proteins in Regulating m(6)A-Modified mRNA[J]. Cell, 2020, 181(7): 1582-95.
13	Wang X, Zhao B, Roundtree I, et al. N6-methyladenosine modulates messenger RNA translation efficiency[J]. Cell, 2015, 161(6): 1388-99.
14	Ma SB, Sun BF, Duan SQ, et al. YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8⁺ T cells[J]. Nat Immunol, 2023, 24(2): 255-66.
15	Huang J, Zhou Y. Emerging role of epigenetic regulations in periodontitis: a literature review[J]. Am J Transl Res, 2022, 14(4): 2162-83.
16	Lagunas-Rangel FA. Fas (CD95)/FasL (CD178) system during ageing[J]. Cell Biol Int, 2023, 47(8): 1295-313.
17	Mahadevan KK, McAndrews KM, LeBleu VS, et al. KRAS(G12D) inhibition reprograms the microenvironment of early and advanced pancreatic cancer to promote FAS-mediated killing by CD8⁺ T cells[J]. Cancer Cell, 2023, 41(9): 1606-20.
18	Chiu HY, Sun GH, Chen SY, et al. Pre-existing fas ligand (FasL) in cancer cells elicits tumor-specific protective immunity, but delayed induction of FasL expression after inoculation facilitates tumor formation[J]. Mol Carcinog, 2013, 52(9): 705-14.
19	Xie YF, Xie F, Zhou XX, et al. Microbiota in tumors: from understanding to application[J]. Adv Sci, 2022, 9(21): e2200470.
20	Sepich-Poore GD, Zitvogel L, Straussman R, et al. The microbiome and human cancer[J]. Science, 2021, 371(6536): eabc4552.
21	Singhrao SK, Harding A, Simmons T, et al. Oral inflammation, tooth loss, risk factors, and association with progression of Alzheimer's disease[J]. J Alzheimers Dis, 2014, 42(3): 723-37.
22	Groeger S, Wu F, Wagenlehner F, et al. PD-L1 up-regulation in prostate cancer cells by Porphyromonas gingivalis [J]. Front Cell Infect Microbiol, 2022, 12: 935806.
23	Mu WX, Jia YQ, Chen XB, et al. Intracellular Porphyromonas gingivalis promotes the proliferation of colorectal cancer cells via the MAPK/ERK signaling pathway[J]. Front Cell Infect Microbiol, 2020, 10: 584798.
24	Liang GF, Wang HJ, Shi H, et al. Porphyromonas gingivalis promotes the proliferation and migration of esophageal squamous cell carcinoma through the miR-194/GRHL3/PTEN/akt axis[J]. ACS Infect Dis, 2020, 6(5): 871-81.
25	Shen L, Zhang D, Gao S. Effect of infection on IFNGR1 palmitoylation in esophageal cancer cells [J].J South Med Uni, 2023, 43: 1155-63.
26	Liu YW, Zhou FY, Yang HJ, et al. Porphyromonas gingivalis promotes malignancy and chemo-resistance via GSK3β‑mediated mitochondrial oxidative phosphorylation in human esophageal squamous cell carcinoma[J]. Transl Oncol, 2023, 32: 101656.
27	Dixit D, Prager BC, Gimple RC, et al. The RNA m6A reader YTHDF2 maintains oncogene expression and is a targetable dependency in glioblastoma stem cells[J]. Cancer Discov, 2021, 11(2): 480-99.
28	Sharma S, Carmona A, Skowronek A, et al. Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95[J]. Nat Commun, 2019, 10(1): 3105.
29	Man SM, Kanneganti TD. Regulation of lysosomal dynamics and autophagy by CTSB/cathepsin B[J]. Autophagy, 2016, 12(12): 2504-5.
30	Debnath J, Gammoh N, Ryan KM. Autophagy and autophagy-related pathways in cancer[J]. Nat Rev Mol Cell Biol, 2023, 24(8): 560-75.
31	Liu JD, Gao MW, Xu SY, et al. YTHDF2/3 are required for somatic reprogramming through different RNA deadenylation pathways[J]. Cell Rep, 2020, 32(10): 108120.
32	Zheng SW, Yu SW, Fan XM, et al. Porphyromonas gingivalis survival skills: immune evasion[J]. J Periodontal Res, 2021, 56(6): 1007-18.
33	Li C, Yu R, Ding YM. Association between Porphyromonas Gingivalis and systemic diseases: focus on T cells-mediated adaptive immunity[J]. Front Cell Infect Microbiol, 2022, 12: 1026457.
34	Xu WZ, Zhou W, Wang HZ, et al. Roles of Porphyromonas gingivalis and its virulence factors in periodontitis[J]. Adv Protein Chem Struct Biol, 2020, 120: 45-84.
35	Yuan X, Liu YW, Kong JY, et al. Different frequencies of Porphyromonas gingivalis infection in cancers of the upper digestive tract[J]. Cancer Lett, 2017, 404: 1-7.
36	阮豪杰, 程维刚, 焦叶林, 等. 菌毛素介导牙龈卟啉单胞菌对食管鳞癌促进作用[J]. 中华微生物学和免疫学杂志, 2022, 42(4): 275-80. DOI: 10.3760/cma.j.cn112309-20210704-00226

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