过表达miR-181a-5p的口腔癌皮下移植瘤小鼠模型的小肠代谢组学研究

A subcutaneous transplanted tumor mouse model of oral cancer overexpressing miR-181a-5p study of small intestine metabolomics

目的通过检测口腔癌皮下移植瘤小鼠小肠中代谢物和代谢通路的变化,分析过表达miR-181a-5p对皮下移植瘤小鼠小肠中代谢物和代谢物组的影响。方法实验分为3组,空白对照(Control)组、阴性对照(negative control,NC)组以及实验(over expression of miR-181a-5p,OE)组。将不同组别处理后的细胞混悬液,通过皮下注射到M-NSG重度免疫缺陷雌性小鼠右侧腹股沟中上部,构建成口腔癌皮下移植瘤小鼠模型。按时记录小鼠体重变化,对小鼠小肠组织进行HE染色,观察各组病理变化。采用超高效液相色谱-串联飞行时间质谱联用仪和串联Orbitrap质谱联用仪检测NC组、OE组和Control组小鼠小肠中的代谢物,使用XCMS预分析原始数据,质量评价样本数据,鉴定Control组与NC组、NC组与OE组的差异代谢物,进行KEGG富集分析获取差异代谢通路。结果Control组和NC组小肠组织中共鉴定出170种差异代谢物。代谢物富集的显著性信号通路包括胆碱代谢、丙氨酸、天冬氨酸和谷氨酸代谢、γ-氨基丁酸(GABA)神经突触代谢、甘油磷脂代谢、环磷酸腺苷(cAMP)信号通路代谢、癌症中心碳代谢以及烟酸和烟碱胺代谢通路。NC组和OE组相比,小鼠小肠中检测到VIP(variable importance in the projection)>2的差异代谢物有16种,显著性差异代谢物包括甘油磷酰胆碱、棕榈酸、3-羟基丁酰肉碱、β-羟丁酸等。代谢物富集到的显著性差异通路为胆碱代谢通路。结论口腔癌皮下移植瘤可引起小鼠小肠的代谢物发生变化,主要改变了小肠中与能量代谢相关的代谢物。过表达miR-181a-5p影响口腔癌皮下移植瘤小鼠小肠代谢物,代谢物富集通路为胆碱代谢通路。

Objective To analyze the effects of miR-181a-5p overexpression on metabolites in the small intestines of mice with subcutaneous oral cancer by detecting changes in metabolites and metabolic pathways.Methods Three groups were included in study:Control group,negative control and miR-181a-5p overexpression group.To establish a subcutaneous oral cancer model in mice,variously treated cell suspensions were subcutaneously injected into the upper right of the groin in female M-NSG severely immunodeficient mice.Changes in pathology and small intestinal tissues were assessed by HE staining.Changes in mouse body weight were also assessed.Tandem orbitrap mass spectrometry and ultrahigh performance liquid chromatography-tandem time-of-flight mass spectrometry,were used to examine metabolites in the small intestines.By pre-analyzing the original data and quality rating sample data,XCMS was able to assess which metabolites were different among the groups.To identify unique metabolic pathways,KEGG enrichment analysis was used.Results A total of 170 distinct metabolites were found in the small intestinal tissues of Control and NC groups.Choline metabolism,alanine,aspartate,and glutamate metabolism,GABA synaptic metabolism,glycerophospholipid metabolism,cAMP signaling route,cancer center carbon metabolism,and niacin and niacin amine metabolic pathways were important signaling pathways for metabolite enrichment.In the NC group,16 distinct metabolites with VIP values larger than 2 were found in the small intestines compared with the OE group overexpressing miR-181a-5p.Glycerin phosphorylcholine,palmitic acid,3-hydroxybutyl carnitine,andβ-hydroxybutyric acid were among the metabolites that significantly varied.The primary enhanced metabolic pathway was the choline pathway.Conclusions Mouse small intestines underwent slight changes from subcutaneous oral cancer with the greatest effect on metabolites critical for energy metabolism.The choline metabolic pathway was the pathway that selected absolutely metabolites in mouse small intestines with subcutaneous grafts of oral cancer.