【摘要】 背景:具核梭杆菌(Fn)是口腔常见的致病菌,研究发现Fn与结直肠癌的发生、发展密切相关,尤其是炎症相关性结直肠癌。目的:探讨Fn感染在结肠癌细胞中形成炎性微环境的机制。方法:构建Fn感染Caco-2细胞的炎症模型,行miRNA测序。将miR-181b mimics或inhibitor转染Fn感染的Caco-2细胞。以qRT-PCR和蛋白质印迹法分别检测TNF-αmRNA和蛋白表达,ELISA法检测上清液中TNF-α含量,Transwell小室法检测穿膜淋巴细胞数。结果:Fn组TNF-αmRNA和蛋白表达均显著高于对照组(P<0.05),上清液中TNF-α含量显著升高(P<0.05),穿膜淋巴细胞数量明显增多(P<0.05)。miRNA测序和qRT-PCR结果均显示,Fn组miR-181b表达较对照组显著降低(P<0.05)。与对照组相比,miR-181b mimics+Fn组TNF-αmRNA和蛋白表达显著降低(P<0.05);而miR-181b inhibitor组TNF-αmRNA和蛋白表达均显著升高(P<0.05)。生物信息学工具和双荧光素酶检测证实TNF-α可能为Caco-2细胞中miR-181b的靶基因。结论:Fn通过抑制Caco-2细胞中miR-181b表达而上调TNF-α表达,募集淋巴细胞形成炎性微环境。更多还原

【Abstract】 Background: Fusobacterium nucleatum( Fn) is a common oral pathogen. Studies have shown that Fn is closely related to the occurrence and development of colorectal cancer,especially the inflammation-related colorectal cancer.Aims: To investigate the mechanism of Fn in forming an inflammatory microenvironment in colon cancer cells. Methods:An inflammation model of Caco-2 cells infected by Fn was constructed,and miRNA sequencing was performed. miR-181 b mimics or inhibitor was transfected into Fn infected Caco-2 cells. mRNA and protein expressions of TNF-α were determined by qRT-PCR and Western blotting,respectively,and concentration of TNF-α in supernatant was measured by ELISA,number of lymphocyte penetrating the membrane was measured by Transwell chamber. Results: Compared with control group,mRNA and protein expressions of TNF-α were significantly increased( P < 0. 05),concentration of TNF-α in supernatant was significantly increased( P < 0. 05),and number of lymphocyte penetrating the membrane was significantly increased in Fn group( P < 0. 05). miRNA sequencing and qRT-PCR results showed that expression of miR-181 b was significantly decreased in Fn group than in control group( P < 0. 05). Compared with control group,mRNA and protein expressions of TNF-α were significantly decreased in miR-181 b mimics + Fn group( P < 0. 05),however,mRNA and protein expressions of TNF-α were significantly increased in miR-181 b inhibitor group( P < 0. 05). Bioinformatics tools and Luciferase assay confirmed that TNF-α might be the target gene of miR-181 b in Caco-2 cells. Conclusions: Fn can upregulate the expression of TNF-α by inhibiting miR-181 b in Caco-2 cells and recruiting lymphocytes to form an inflammatory microenvironment.更多还原