牙龈卟啉单胞菌通过xCT受体调控食管癌细胞谷氨酰胺代谢

Study on the Mechanism of Porphyromonas gingivalis Regulating the Metabolism of Esophageal Cancer Through xCT Receptors

目的探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)通过膜受体分子-溶质运载蛋白7家族成员11(solute cartier family 7 member 11,SLC7A11,又称xCT)调节食管鳞癌细胞谷氨酰胺代谢的作用及机制。方法基于人类全基因组库筛选Pg感染食管鳞癌细胞所需的宿主因子;运用免疫共沉淀、NanoBiT活细胞实时蛋白相互作用和免疫荧光实验探索Pg的主要毒力因子鞭毛蛋白(fimbriae,FimA)和宿主细胞受体分子xCT之间的相互作用关系;构建xCT基因敲除小鼠模型,Pg口腔灌胃4周后剥离小鼠食管组织,利用RNAscope原位杂交检测小鼠食管黏膜Pg的载菌量;采用qRT-PCR、Western blot和还原型/氧化型谷胱甘肽试剂盒测定Pg感染对食管癌细胞内谷氨酰胺代谢及其合成酶表达的影响。结果全基因组筛选测序分析及细胞活力测定结果表明,xCT能够增强Pg在胞内的定殖从而促进食管癌细胞增殖;免疫共沉淀、NanoBiT实验和免疫细胞荧光实验结果表明,xCT与Pg鞭毛蛋白FimA可直接结合并相互作用;体内实验结果表明,xCT基因敲除小鼠食管上皮中Pg载菌量显著低于野生型小鼠(P<0.01);qRT-PCR、Western blot和还原型/氧化型谷胱甘肽测定发现,感染Pg后食管癌细胞的谷氨酰胺合成酶(glutaminase-1,GLS1)以及还原型/氧化型谷胱甘肽比率显著高于未感染组,差异具有统计学意义(均P<0.01)。结论xCT是Pg入侵食管癌细胞的重要受体分子之一;Pg入胞后能显著增强肿瘤细胞谷氨酰胺代谢。上述研究为食管癌的病因学和防治策略提供了理论依据。

Objective To investigate regulative effect of Porphyromonas gingivalis(Pg)on glutamine metabolism in esophageal squamous cell carcinoma cells through membrane receptor molecule-solute cartier family 7 member 11(SLC7A11,xCT).Methods The host factors required by Pg to infect esophageal squamous cell carcinoma(ESCC)cells were screened from the human whole genome library.Co-immunoprecipitation,NanoBiT live cell real-time protein interaction and immunofluorescence experiments were used to explore the interaction relationship between the main virulence factor fimbriae(FimA)of Pg and the host cell receptor molecule xCT.The xCT gene knockout mouse model was constructed,and the mouse esophagus tissue was excised after oral administration of Pg for 4 weeks.The colonization of Pg in the mouse esophageal mucosa was detected by RNAscope in situ hybridization.The effects of Pg infection on glutamine metabolism and its synthetase expression in ESCC cells were determined by qRT-PCR,Western blot and oxidized glutathione kits.Results The results of whole genome-wide screening and sequencing analysis as well as cell viability assay showed that xCT could enhance the intracellular colonization of Pg to promote the proliferation of ESCC cells.The Co-immunoprecipitation,NanoBiT experiments and immunocytofluorescence experiments showed that xCT could directly bind and interact with Pg-derived FimA.In vivo experiments showed that the bacterial load of Pg in the esophageal epithelium of xCT knockout mice was significantly lower than that of wild-type mice(P<0.01).qRT-PCR,Western blot,and reduced/oxidized glutathione assays found that the glutamine synthetase(glutaminase-1,GLS1)and the ratio of glutathione/glutathione-oxidized in ESCC cells after infection with Pg were significantly higher than those in the infected group,and the differences were statistically significant(all P<0.05).Conclusion xCT is one of the important receptor molecules of Pg to invade ESCC cells.Pg can significantly enhance the glutamine metabolism of tumor cells after colonization in ESCC cells.This study provides theoretical basis for study of the etiology and prevention strategies of esophageal cancer.