目的:探讨牙龈卟啉单胞菌(Pg)感染对食管癌细胞免疫逃逸的影响及YTHDF2和Fas在此调控机制中的作用。
方法:我们使用免疫组织化学方法检测了有和没有Pg感染的食管鳞状细胞癌(ESCC)组织中的YTHDF2和Fas蛋白表达,并使用Western印迹方法检测了Pg感染的KYSE150细胞。通过免疫共沉淀(Co-IP)研究YTHDF2和Fas之间的相互作用。用慢病毒介导的YTHDF2敲低的Pg感染的KYSE150细胞检查YTHDF2,组织蛋白酶B(CTSB)的表达水平的变化,Fas和FasL蛋白,并且观察到E64(一种组织蛋白酶抑制剂)对这些蛋白质的作用。在Pg感染和E64治疗后,KYSE150细胞与人外周血单核细胞(PBMC)共培养,流式细胞术检测T细胞相关效应分子的表达。
结果:ESCC组织和Pg感染细胞显示YTHDF2表达显著增加,Fas表达降低。Co-IP的结果证明了YTHDF2和Fas之间的直接相互作用。在YTHDF2敲低的Pg感染的KYSE150细胞中,CTSB表达显著降低,Fas和FasL表达显著升高。E64处理KYSE150细胞显著降低CTSB的表达而不影响YTHDF2的表达,显著增加Fas和FasL的表达。流式细胞术显示,在与PBMC共培养的Pg感染KYSE150细胞中,颗粒酶B和Ki67的表达显著降低,而PD-1的表达显著增强。
结论:Pg感染YTHDF2依赖性调节Fas的表达,促进食管癌的免疫逃逸,从而促进癌症进展,提示YTHDF2在调节食管癌免疫逃逸中的关键作用。
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.
CONCLUSIONS: 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.