Fusobacterium Nucleatum Potentiates Intestinal Tumorigenesis In Mice Via A Toll-like Receptor 4/P21-activated Kinase 1 Cascade

Objective: The development of colorectal cancer is related to environment,gene,microorganism and other factors.In recent years,a large number of studies have shown that gut micribiota and colorectal cancer(CRC)have a very complex relationship,includes: fusobacterium nucleatum,Escherichia coli,fragile bacillus,and Bacteroides fragilis especially with fusobacterium nucleatum(Fn).Fn is a kind of gram-negative obligate anaerobe that is common parasitic in the oral cavity and associated with many diseases.It is the main pathogenic bacteria of periodontitis,still can cause premature birth,cardiovascular disease,rheumatism,etc.A large number of studies have shown that there is a rich Fn in the precancerous lesion and cancer tissue of colorectal cancer compared to the adjacent tissues or normal colonic tissues adjacent to CRC.Fn colonized in the gut or cancer tissue can promote the release of inflammatory factors produce inflammation environment,inhibit cancer related activity of lymphocytes,weaken the antitumor immune ability,activate the cancer related signaling pathways,and other complex mechanism to promote the development of CRC.Fn,for example,as a kind of gram-negative bacteria,the bacterial endotoxin can also be a strong pathogenic factor,there are many evidence proved that Fn can activate MYD88 dependence of TLR4 signaling pathways,inducing the activation of nuclear factor NF-B ? and microRNA21 expression level increased,thus promotes the proliferation of tumor cells and inflammation related to the occurrence of colon cancer,and Fn can be caused by activation of beta-catenin,C-myc and the increased expression of cyclinD1,thus promote the occurrence of CRC,PAK1 is an important protein in CRC,can activate the beta-catenin(scr675)signaling pathway to promote the occurrence of CRC.However,little is known aboutthe potential specific pathogenesis of Fn in the occurrence of colorectal cancer(CRC).This experiment uses animal models to explore the potential mechanism of Fn promoting CRC.Methods: select 25 wild type C57 BL / 6mice and 25 C57 BL / 6-ApcMin / + mice,then randomly and equably divided into five groups,respectively: control group(Ctr),,antibiotic treatment group(Ctr+Ab),antibiotics and Fn group(Fn+Ab),antibiotics and Fn and TAK-242-4(toll-like receptors antagonists)group(Fn+Ab+T),antibiotics and Fn and DMSO group(Fn+Ab+D).We studied the effects of antibiotics,Fn and TAK-242 on the structure of gut microbiota and intestinal tumor formation in mice.We used high-throughput sequencing to observe the changing structure of the gut microbiota in each group,naked eye and HE staining to determine pathological properties of intestinal tumors in mice,FISH to observe whether there is a Fn invade the intestinal mucosa.In addition,we also use of immunohistochemistry and western blot method to detect the expression of toll-like 4(TLR4),p21-activated kinase 1(PAK1),phosphorylated-PAK1(p-PAK1)and phosphorylated-beta-catenin S675(p-beta-catenin S675)and the cell cycle protein D1(cyclinD1)among groups in intestinal tumors of mice.In order to verify our conjecture: Fn via TLR4 / p-PAK1 pathway to promote intestinal tumor development.Results: the treatment of Fn and antibiotics changed the microorganism structure of the gut in mice,and Fn could increase the abundance of opportunistic pathogens and reduce the abundance of probiotics,such as the obvious increase of Fn.In addition,In addition,Fn invaded into the intestinal mucosa in large amounts but were less abundant in the feces of Fn-fed mice,but the abundance of feces in the colon of mice is lower,which may be related to the invasiveness of Fn.moreover,in ApcMin /+ mice,can see clearly observed the tumor formation,HE staining can also be observed in tumor tissue,compared with control group,The average number and size of intestinal tumors in Fn groups was significantly increased compared to control groups in ApcMin/+ mice(P < 0.05).At the same time,compared with the isolated Fn and antibiotic treatment group,the average number and average size of the intestinal tumors in the mice were significantly reduced after TAK-242 treatment.Fn can up-regulate the expression of TLR4,PAK1,p-pak1,p-beta-catenin S675 and cyclinD1 in cancer tissues(p <0.05).In addition,compared with Fn,TAK-242 significantly reduced the average number and size of intestinal tumors(P < 0.05).The expression of p-pak1,p-beta-catenin S675,and cyclin D1 was significantly reduced(P <0.05).In wild type C57 BL / 6 mice,although there is no obvious tumor formation was observed,Significant Fn invasion was observed in the intestinal mucosa,the expression of TLR4,PAK1,p-PAK1,p-beta-catenin S675 and cyclinD1 trend is consistent with the mutant mice.Conclusions: Fn potentiates intestinal tumorigenesis in ApcMin/+ mice via a TLR4/p-PAK1/p-?-catenin S675 cascade.Fn-induced intestinal tumorigenesis can be inhibited by TAK-242,implicating TLR4 as a potential target for the prevention and therapy of Fn-related CRC.