牙龈卟啉单胞菌感染对食管癌细胞内IFNGR1棕榈酰化位点突变的影响

Effect of Porphyromonas gingivalis infection on IFNGR1 palmitoylation in esophageal cancer cells

目的探讨牙龈卟啉单胞菌(Pg)感染对食管鳞癌细胞(ESCC)内IFNGR1棕榈酰化位点突变的机制及临床意义。方法Western blot检测Pg感染不同时间(24、48 h)ESCC细胞KYSE30及KYSE70中IFNGR1蛋白量表达水平。2-BP检测IFNGR1的棕榈酰化作用。构建IFNGR1-WT和IFNGR1-C122A棕榈酰化位点突变位点质粒,建立IFNGR1-WT和IFNGR1-C122A稳定细胞系,分别用Pg感染IFNGR1-WT和IFNGR1-C122A细胞,并分为4组:IFNGR1-WT、IFNGR1-C122A、IFNGR1-WT+Pg和IFNGR1-C122A+Pg组。免疫荧光和click-it实验检测IFNGR1在122位点发生棕榈酰化,以及Pg促进IFNGR1在ESCC内发生棕榈酰化。利用平板克隆、划痕实验及Transwell法检测Pg感染前后,IFNGR1-WT和IFNGR1-C122A稳转细胞株的体外增殖、迁移与侵袭能力差异。免疫荧光实验检测IFNGR1与溶酶体标记物LAMP2共定位情况。免疫组化检测50例ESCC组织中Pg感染与IFNGR1蛋白的表达情况;采用卡方检验及R Studio软件分析二者与ESCC患者临床病理特征及生存预后之间的相关性。结果Pg感染ESCC下调IFNGR1的蛋白表达。IFNGR1在122位点发生棕榈酰化。Pg促进IFNGR1在ESCC内棕榈酰化。与IFNGR1-WT组相比,IFNGR1-WT+Pg组体外增殖253±6.245 vs 52±2.45、迁移(0.7816±0.0071)%vs(0.4347±0.0366)%及侵袭709.33±14.57 vs 356.3±17.39能力均显著增强(P<0.05);与IFNGR1-C122A组相比,IFNGR1-C122A+Pg组细胞体外增殖137.33±4.726 vs 29.67±3.055、迁移(0.7477±0.0057)%vs(0.2406±0.0028)%及侵袭587.33±5.033 vs 67.33±2.517能力同样均显著增强(P<0.05)。与IFNGR1-WT+Pg组相比,IFNGR1-C122A+Pg体外增殖137.33±4.726 vs 253±6.245、迁移(0.7477±0.0057)%vs(0.7816±0.0071)%及侵袭587.33±5.033 vs 709.33±14.57能力均显著降低(P<0.05)。Pg及ZDHHC3促进IFNGR1在溶酶体内降解;IFNGR1与Pg表达呈负性相关,且IFNGR1的表达降低与更差的病理特征及生存预后相关。结论Pg感染ESCC下调IFNGR1蛋白表达量,同时诱导IFNGR1棕榈酰化,促进ESCC恶性化进展;IFNGR1的棕榈酰化位点突变减少ESCC增殖、迁移和侵袭作用;Pg感染ESCC靶向IFNGR1到溶酶体内降解可能是由于棕榈酰化作用,因此,清除Pg或抑制棕榈酰化作用能有效抑制食管癌的恶性进展。

Objective To investigate the effect of Porphyromonas gingivalis(Pg)infection on IFNGR1 palmitoylation and biological behaviors of esophageal squamous cell carcinoma(ESCC)cells and the clinical implications.Methods The expression levels of IFNGR1 protein in ESCC cell lines KYSE30 and KYSE70 were detected using Western blotting at 24 and 48 h after Pg infection,and 2-BP was used to detect IFNGR1 palmitoylation in the cells.KYSE70 cells with wild-type IFNGR1(IFNGR1-WT cells)and with IFNGR1-C122A palmitoylation site mutation induced by site-specific mutagenesis(IFNGR1-C122A cells)were both infected with Pg,and the changes in palmitoylation of IFNGR1-C122A were analyzed using immunofluorescence and Click-iT assays.The changes in proliferation,migration and invasion ability of the infected cells were evaluated using plate cloning assay,scratch assay and Transwell assay,and IFNGR1 co-localization with lysosomal marker LAMP2 was dected using immunofluorescence assay.Immunohistochemistry was used to detect Pg infection and IFNGR1 protein expression in 50 ESCC tissues,and their correlation with the clinicopathological characteristics and survival outcomes of the patients was analyzed.Results Pg infection down-regulated the protein expression of IFNGR1 in ESCC and promoted IFNGR1 palmitoylation at site 122.In IFNGR1-WT cells,Pg infection significantly enhanced cell proliferation,migration and invasion(P<0.05).Similarly,Pg also significantly promoted proliferation,migration and invasion of IFNGR1-C122A cells,but to a lesser extent as compared with the wild-type cells(P<0.05).Immunofluorescence assay showed that Pg and ZDHHC3 promoted IFNGR1 degradation within the lysosome.Immunohistochemical studies of the ESCC tissue samples showed a negative correlation between IFNGR1 and Pg expression,and a reduced IFNGR1 expression was correlated with a poorer survival outcome of the patient.Conclusion Pg infection enhances IFNGR1 palmitoylation to promote progression of ESCC,and elimination of Pg and inhibiting IFNGR1 palmitoylation may effectively control ESCC progression.