【作者】 张涛；

【导师】 曾冬竹；

【作者基本信息】 重庆医科大学 ， 外科学， 2024， 硕士

【摘要】 研究背景与目的结直肠癌(colorectal cancer,CRC)是人类最常见的消化系统肿瘤之一,而结直肠癌转移(colorectal cancer metastases,CRM)是CRC患者死亡的主要原因。研究表明,肿瘤微环境组成状态的改变与结直肠癌的发生发展及转移密切相关,而具核梭杆菌和巨噬细胞是结直肠癌肿瘤微环境的重要组成部分。具核梭杆菌(Fusobacterium nucleatum,F.nucleatum)是一种常见的口腔革兰氏阴性厌氧菌,越来越多的证据表明其在结直肠癌的发展、转移和化疗耐药性中发挥作用。巨噬细胞(macrophage,Mφ)是肿瘤微环境中的主要免疫细胞,它们可以分化为M1和M2两种不同的亚型。M1亚型以炎症响应为主要特征,有助于抵抗感染和激活免疫反应;相对地,M2亚型则以抗炎作用为主,能够抑制T细胞的活性,并且有助于肿瘤的生长、侵袭和转移。研究发现,F.nucleatum感染能够增加肿瘤相关巨噬细胞(Tumor-associated macrophages,TAM)的数量,并且促使这些巨噬细胞向M2型转化,从而促进结直肠癌的发展。然而,关于F.nucleatum如何促进巨噬细胞M2极化的分子机制还有待进一步阐明。研究表明,JAK/STAT信号途径对于M1/M2巨噬细胞的极化起着决定性的作用,其中STAT6蛋白是M2型肿瘤相关巨噬细胞(M2-TAMs)活化过程中的核心要素。在JAK被激活之后,STAT6会形成磷酸化二聚体的形式,进而转移到细胞核内,从而调节与M2-TAMs相关基因的表达。此外,氧连接N-乙酰葡萄糖胺(O-linked N-acetylglucosamine,O-GlcNAc)糖基化修饰也具有增强M2-TAMs相关基因转录活性的作用。O-GlcNAc糖基化修饰由Hart与Torres于1984年发现,它与传统的蛋白糖基化修饰最大的区别是仅于蛋白质的丝氨酸/苏氨酸残基上加成一个单糖。O-GlcNAc糖基化修饰主要存在于细胞质和细胞核,由于其能快速、可逆的添加及移除,能可逆的竞争某些磷酸化修饰,迅速调节蛋白质的活性,几乎参与了各种细胞代谢途径和信号转导通路,在肿瘤和糖尿病等多种疾病中起着非常重要的调节作用。研究表明,F.nucleatum能够增强结直肠癌细胞的糖酵解活性从而促进CRC细胞增殖和转移。由于糖酵解的主要代谢产物UDP-N-乙酰葡糖胺(UDP-GlcNAc)正是O-GlcNAc糖基化修饰的必要底物,因此我们推测具核梭杆菌也可能促进巨噬细胞糖酵解从而增强巨噬细胞O-GlcNAc糖基化修饰和M2极化。然而,F.nucleatum能否通过促进巨噬细胞糖酵解和O-GlcNAc糖基化修饰而增强M2型极化,目前尚无报道。本研究旨在探讨F.nucleatum与巨噬细胞M2极化在结直肠癌转移中的作用及其分子机制。通过生物信息学分析、体外共培养和免疫共沉淀实验,我们证实了F.nucleatum感染通过增强巨噬细胞对葡萄糖的摄取从而促进巨噬细胞内STAT6的O-GlcNAc糖基化修饰,进而诱导巨噬细胞M2型极化最终促进CRC转移。方法1.F.nucleatum感染通过增强STAT6 O-GlcNAc糖基化修饰诱导巨噬细胞M2型极化1.1 F.nucleatum感染对巨噬细胞极化的影响1)F.nucleatum感染RAW264.7细胞24小时后,qPCR检测M1型巨噬细胞的基因包括i NOS和TNF-α,M2型巨噬细胞的基因包括Arg-1和CD206;Western blot检测M1型巨噬细胞的标志蛋白CD86和M2型巨噬细胞的标志蛋白CD206;1.2 F.nucleatum促进STAT6的糖基化修饰1)通过在线数据分析预测STAT6的糖基化修饰位点;2)Western blot检测F.nucleatum促进巨噬细胞O-GlcNAc糖基化修饰情况,以及STAT6蛋白质表达;1.3 STAT6的糖基化修饰促进巨噬细胞M2极化1)F.nucleatum感染巨噬细胞后,Western blot检测OGT和OGA蛋白质及转录水平的变化;2)当敲降OGT再感染F.nucleatum后,Western blot检测RAW264.7细胞O-GlcNAc糖基化修饰的情况,以及STAT6蛋白质表达;2.F.nucleatum感染通过上调GLUT1促进巨噬细胞M2极化和结直肠癌细胞迁移2.1 F.nucleatum对巨噬细胞葡萄糖代谢的影响1)F.nucleatum感染RAW264.7巨噬细胞24小时后,使用2-脱氧葡萄糖荧光类似物(2-NBDG)处理1小时,再通过荧光酶标仪检测巨噬细胞对葡萄糖的摄取能力;2)在RAW264.7细胞中敲降GLUT1后,F.nucleatum感染24小时,再使用2-NBDG处理后检测巨噬细胞对葡萄糖的摄取能力;3)在F.nucleatum感染RAW264.7细胞后,qPCR和Western blot分别检测GLUT1、GLUT2、GLUT3和GLUT4的mRNA和蛋白水平;4)过表达GLUT1后,Western blot检测STAT6的O-GlcNAc糖基化修饰。2.2 F.nucleatum通过GLUT1促进巨噬细胞M2极化和结直肠癌细胞迁移1)F.nucleatum的间接作用:F.nucleatum感染RAW264.7细胞24 h后,将培养基上清制成相关条件培养基[(Mφ+Fn)-CM],用该条件培养基孵育CRC细胞24小时,检测CRC细胞增殖及迁移能力变化;2)机制探讨:转染siGLUT1到RAW264.7细胞24 h、再经F.nucleatum处理24小时后,收集培养基上清液,制备成条件培养基[(Mφ+siGLUT1+Fn)-CM],用该CM孵育CRC细胞后,分别检测其迁移能力的变化;结果1.F.nucleatum感染通过增强STAT6 O-GlcNAc糖基化修饰诱导巨噬细胞M2型极化1.1 F.nucleatum促进巨噬细胞向M2型极化F.nucleatum处理的RAW264.7巨噬细胞:1)M1型巨噬细胞标志基因i NOS和TNF-α的mRNA水平表达降低(P<0.05或0.001),M2型巨噬细胞的标志基因Arg-1和CD206的mRNA水平升高(P<0.05或0.001);2)与对照组相比,F.nucleatum感染组的CD86表达降低,而CD206表达显著升高。提示,F.nucleatum促进RAW264.7细胞向M2型巨噬细胞极化,即形成M2型肿瘤相关巨噬细胞。1.2 F.nucleatum促进STAT6的糖基化修饰1)通过在线数据分析,预测到STAT6具有O-GlcNAc糖基化修饰位点;2)F.nucleatum感染组与PBS对照组相比,RAW264.7细胞的STAT6的O-GlcNAc糖基化修饰水平显著高于相应的对照组。提示:F.nucleatum促进RAW264.7细胞STAT6的O-GlcNAc糖基化修饰。1.3 STAT6的糖基化修饰促进巨噬细胞的M2型极化siOGT+Fn感染组的STAT6的M1型巨噬细胞的标志基因i NOS和TNF-α的mRNA水平与PBS对照组相比明显降低,M2型巨噬细胞的标志基因IL-10和CD206的mRNA水平与PBS对照组相比明显升高;F.nucleatum感染组的CD86表达水平降低、CD206表达水平升高。提示:F.nucleatum通过STAT6的O-GlcNAc糖基化修饰促进巨噬细胞的M2型极化。2.F.nucleatum感染通过上调GLUT1促进巨噬细胞STAT6 O-GlcNAc糖基化修饰从而增强结直肠癌细胞迁移2.1 F.nucleatum促进巨噬细胞O-GlcNAc糖基化修饰和GLUT1表达1)荧光酶标仪读数检测RAW264.7细胞摄取2-NBDG的能力实验结果表明,F.nucleatum感染显著增强巨噬细胞对葡萄糖的摄取能力。q RT-PCR结果显示,F.nucleatum感染显著促进葡萄糖转运体GLUT1和GLUT3的mRNA表达;Western blot结果提示,F.nucleatum感染显著增加葡萄糖转运体GLUT1的蛋白水平,而GLUT2、GLUT3和GLUT4的蛋白表达无显著变化;2)F.nucleatum对RAW264.7巨噬细胞O-GlcNAc糖基化修饰影响。实验结果:F.nucleatum感染组巨噬细胞的O-GlcNAc糖基化修饰水平显著增高,且糖基化修饰转移酶OGT的mRNA水平和蛋白质水平高于对照组(P<0.05),而OGA的mRNA水平和蛋白质水平同PBS对照组相比无明显差异。提示F.nucleatum感染可上调RAW264.7细胞中O-GlcNAc糖基化修饰水平。2.2 F.nucleatum通过GLUT1促进巨噬细胞M2极化和结直肠癌细胞迁移与PBS对照组相比,(Mφ+Fn)-CM组的RKO结直肠癌细胞的迁移能力明显增强,(Mφ+siGLUT1+Fn)-CM组中细胞的迁移能力显著低于(Mφ+siNC+Fn)-CM对照组。上述结果提示,F.nucleatum感染通过GLUT1促进RAW264.7巨噬细胞M2极化间接促进CRC细胞迁移。结论1.F.nucleatum感染促进巨噬细胞向M2型极化,其机制涉及STAT6的O-GlcNAc糖基化修饰2.F.nucleatum感染通过上调GLUT1促进巨噬细胞M2极化和结直肠癌细胞迁移更多还原

【Abstract】 Background and objectiveColorectal cancer(colorectal cancer,CRC)is the most common gastrointestinal tumor,and colorectal cancer metastasis(colorectal cancer metastases,CRM)is the main cause of death in CRC patients.Studies have shown that the change of the composition of the tumor microenvironment is closely related to the development and metastasis of colorectal cancer,and F.nucleatum and macrophages are important components of the tumor microenvironment of colorectal cancer.The Fusobacterium nucleatum(F.nucleatum)is a common oral gram-negative anaerobic bacterium,with increasing evidence for a role in colorectal cancer development,metastasis,and chemoresistance.Macrophages(macrophage,Mφ)are the most abundant infiltrating immune cells in the tumor microenvironment,which can be polarized into M1 and M2.M1 subtype is characterized by inflammatory response,which helps to resist infection and activate immune response.In contrast,M2 subtype is mainly anti-inflammatory,which can inhibit the activity of T cells and contribute to the growth,invasion and metastasis of tumors.F.nucleatum infection was found to increase tumor-associated macrophage(Tumor-associated macrophages,TAM)infiltration and was involved in mediating M2 polarization and promoting CRC progression.However,the molecular mechanisms of how F.nucleatum promotes macrophage M2 polarization remain to be clarified.It shows that JAK/STAT signaling plays a key role in M1/M2 type macrophage polarization,and STAT6 is a key factor in the activation of M2-TAMs.When JAK is activated,phosphorylated STAT6 enters the nucleus,thereby regulating the expression of genes associated with M2-TAMs.In addition,oxygen-linked N-acetylglucosamine(O-linked N-acetylglucosamine,O-GlcNAc)glycosylation modification also enhances the transcriptional activity of M2-TAMs related genes.The O-GlcNAc glycosylation modification was discovered by Hart and Torres in 1984,and the biggest difference from the traditional protein glycosylation modification is the addition of a monosaccharide only to the serine/threonine residue of the protein.O-GlcNAc glycosylation modification mainly exists in the cytoplasm and nucleus.Because it can be added and removed rapidly and reversibly,it can compete with some phosphorylation modifications reversibly,and quickly regulate the activity of proteins.It is almost involved in various cellular metabolic pathways and signal transduction pathways,and plays a very important regulatory role in many diseases,such as tumor and diabetes.It is shown that F.nucleatum is able to enhance the glycolytic activity of colorectal cancer cells and thereby promote CRC cell proliferation and the metastasis.Since the main metabolite of glycolysis,UDP-N-acetylglusamine(UDP-GlcNAc),is the essential substrate for O-GlcNAc glycosylation,we speculated that F.nucleatum would also promote glycolysis in macrophages to enhance O-GlcNAc glycosylation and M2polarization in macrophages.However,there was no report on whether F.nucleatum could enhance M2 type polarization by promoting glycolysis and O-GlcNAc glycosylation modifications in macrophages.This study aimed to investigate the role and molecular mechanism of F.nucleatum and macrophage M2 polarization in colorectal cancer metastasis.Through bioinformatics analysis,in vitro co-culture and immunoprecipitation experiments,we confirmed that F.nucleatum infection promoted O-GlcNAc glycosylation modification of STAT6 in macrophages by enhancing glucose uptake in macrophages,eventually inducing macrophage M2 type polarization and ultimately promoting colorectal cancer metastasis.Methods1.F.nucleatum infection enhances STAT6 O-GlcNAc glycosylation modification and thus promotes M2 polarization of macrophages1.1 F.nucleatum promotes macrophage polarizationAfter 24 hours of F.nucleatum infection in RAW264.7 cells,qPCR was performed to detect the gene expression of M1 macrophage markers including i NOS and TNF-α,as well as M2 macrophage markers including Arg-1 and CD206.Western blot analysis was conducted to assess the protein expression of CD86 as a marker for M1 macrophages and CD206as a marker for M2 macrophages.1.2 F.nucleatum promotes glycosylation modification of STAT6;1)Prediction of glycosylation modification site of STAT6 using bioinformatics software;2)Detection of glycosylation modification of macrophage by western blot with F.nucleatum infection in RAW264.7 cells.Co-immunoprecipitation experiments were performed with STAT6 and s WGA antibodies,and western blot detected the protein expression of STAT6 and O-GlcNAc glycosylation modification;1.3 Glycosylation modification of STAT6 promoted M2 type polarization of macrophages1)Following F.nucleatum infection of macrophages,Western blot analysis was performed to examine the changes in protein and transcript levels of OGT and OGA.2)After suppressing the expression of OGT in RAW264.7 cells,followed by a 24-hour infection with F.nucleatum,the expression levels of total O-GlcNAcylation modification and O-GlcNAcylation modification of STAT6 protein were subsequently evaluated using Western blot analysis in RAW264.7 cells.2.F.nucleatum promotes M2 polarization of macrophages and migration of colorectal cancer cells by upregulating the expression of GLUT1.2.1 F.nucleatum affects the glucose metabolism of macrophages.1)Initially,RAW264.7 macrophages were infected with F.nucleatum for24 hours.Subsequently,the cells were treated with 2-deoxyglucose fluorescent analog(2-NBDG)for 1 hour.Finally,the glucose uptake capacity of the macrophages was assessed using a fluorescence microplate reader.2)The expression of GLUT1 was inhibited in RAW264.7 cells,followed by a 24-hour infection with F.nucleatum.Finally,the cells were treated with 2-deoxyglucose fluorescent analog(2-NBDG),and the glucose uptake capacity of macrophages was assessed using a fluorescence microplate reader.3)F.nucleatum was used to infect RAW264.7 cells for 24 hours.Subsequently,qPCR and Western blot techniques were employed to respectively examine the mRNA and protein expression levels of GLUT1,GLUT2,GLUT3,and GLUT4.4)After overexpressing GLUT1 in RAW264.7 cells,the O-GlcNAcylation modification level of STAT6 was detected using Western blot analysis.2.2 F.nucleatum promotes M2 polarization of macrophages and facilitates colorectal cancer cell migration through GLUT1.1)Indirect effect of F.nucleatum:After infecting RAW264.7 cells with F.nucleatum for 24 hours,the conditioned medium(CM)was collected and prepared as the relevant conditioned medium[(Mφ+Fn)-CM].CRC cells were then incubated with this conditioned medium for 24hours to assess changes in CRC cell proliferation and migration abilities.2)Mechanistic exploration:After transfecting siGLUT1 into RAW264.7 cells for 24 hours,followed by treatment with F.nucleatum for an additional 24 hours,the conditioned medium(CM)was collected and prepared as the relevant conditioned medium[(Mφ+siGLUT1+Fn)-CM].CRC cells were then incubated with this CM,and changes in their migration ability were assessed.Results1.F.nucleatum infection enhanced STAT6 O-GlcNAcylation modification to induce M2 polarization of macrophages.1.1 F.nucleatum promoted polarization of macrophages towards the M2 phenotype.In F.nucleatum-infected RAW264.7 macrophages:1)The mRNA expression levels of M1 macrophage marker genes i NOS and TNF-αwere decreased(P<0.05 or 0.001),while the mRNA expression levels of M2 macrophage marker genes Arg-1 and CD206were increased(P<0.05 or 0.001).2)Compared to the control group,the F.nucleatum-infected group showed decreased expression of CD86 and significantly increased expression of CD206.These findings suggested that F.nucleatum promoted polarization of RAW264.7 cells towards the M2 phenotype,thereby forming M2 tumor-associated macrophages.1.2 F.nucleatum promoted the glycosylation modification of STAT6.1)Through online data analysis,putative O-GlcNAcylation modification sites were predicted in STAT6.2)Compared to the PBS control group,the F.nucleatum-infected group showed a significantly higher level of O-GlcNAcylation modification of STAT6 in RAW264.7 cells.This suggested that F.nucleatum promoted O-GlcNAcylation modification of STAT6 in RAW264.7 cells.1.3 The glycosylation modification of STAT6 promoted the M2polarization of macrophages.In the siOGT+Fn-infected group,the mRNA levels of M1 macrophage marker genes i NOS and TNF-αwere significantly decreased compared to the PBS control group,while the mRNA levels of M2macrophage marker genes IL-10 and CD206 were significantly increased compared to the PBS control group.The F.nucleatum-infected group showed decreased expression of CD86 and significantly increased expression of CD206.These findings suggested that F.nucleatum promoted M2 polarization of macrophages through O-GlcNAcylation modification of STAT6.2.F.nucleatum infection enhanced colorectal cancer cell migration by upregulating GLUT1 to promote O-GlcNAcylation modification of STAT6 in macrophages.2.1 F.nucleatum promoted O-GlcNAcylation modification and GLUT1 expression in macrophages.1)Fluorescence spectrophotometer readings were used to assess the uptake capacity of 2-NBDG in RAW264.7 cells,and the experimental results demonstrated that F.nucleatum infection significantly enhanced macrophage glucose uptake ability.q RT-PCR results showed that F.nucleatum infection significantly promoted the mRNA expression of glucose transporters GLUT1 and GLUT3.Western blot results indicated that F.nucleatum infection significantly increased the protein level of glucose transporter GLUT1,while the protein expression of GLUT2,GLUT3,and GLUT4 showed no significant change.2)F.nucleatum impacted O-GlcNAcylation modification in RAW264.7 macrophages.Experimental results showed that the level of O-GlcNAcylation modification in macrophages was significantly increased in the F.nucleatum-infected group,and the mRNA and protein levels of the glycosylation transferase OGT were higher than the control group(P<0.05).However,there were no significant differences in the mRNA and protein levels of OGA compared to the PBS control group.These findings suggested that F.nucleatum infection could upregulate the level of O-GlcNAcylation modification in RAW264.7 cells.2.2 F.nucleatum promoted M2 polarization of macrophages and colorectal cancer cell migration through GLUT1.Compared to the PBS control group,the migration ability of RKO colorectal cancer cells was significantly enhanced in the(Mφ+Fn)-CM group,while the migration ability of cells in the(Mφ+siGLUT1+Fn)-CM group was significantly lower than the(Mφ+si NC+Fn)-CM control group.These results suggested that F.nucleatum infection indirectly promoted CRC cell migration through GLUT1 by promoting M2 polarization of RAW264.7 macrophages.Conclusion1.F.nucleatum promotes macrophage polarization from macrophages to M2 type.Its mechanism involves O-GlcNAc glycosylation modification of STAT 6.2.F.nucleatum infection promotes macrophage M2 polarization and colorectal cancer cell migration,through upregulation of GLUT 1更多还原