Study Of Deoxycholic Acid-Mediated-Ggut Dysbiosis Induced Intestinal Adenoma-Adenocarcinoma Sequence

Aims Colorectal cancer is one of the most common tumor in the worldwide. The mutation of adenomatous polyposis coli gene is an early event among the multistep progress of colorectal cancer. Studies have shown high fat diet can alter the structure of intestinal microbiota, and also increase the concentration of deoxycholic acid（DCA）, a secondary bile acid in the intestinal lumen. Evidence has been demonstrated that intestinal carcinogenesis is significantly associated with luminal continuously burden of DCA. However, the causality between intestinal microbiota and tumorigenesis is still remained to be clarified.Methods Part 1 Four weeks old wild-type mice and Apc^min/+ mice were randomized into three groups: wild-type group（WT group, 0.2% DCA in drinking water）, Apc^min/+ control group（ordinary drinking water） and Apc^min/+ DCA group（DCA group）. Feces were collected at week 0 and week 12, then analysed by 16 S r RNA pyrosequencing. All the mice were sacrificed at week 12. Intestinal tumor development were evaluated by microscope and HE staining. Immunohistochemistry（IHC）, and TUNEL were used to detect cell proliferation and apoptosis. Proteins of intestinal tight junctions were detected by immunofluorescence and real-time PCR. Intestinal permeability was assessed by concentration of FITC-D in serum. Part 2 To further determine the causality between gut microbiota and intestinal carcinogenesis, fecal donors were assigned to the control and DCA group, which were stopped to have 2 weeks’ washout period. Mice were administrated of different fecal fluid and divided into 3 groups: FMT 1 group（from DCA group）, FMT 2 group（from control group） and FMT 3 group（from DCA group）. Mice received streptomycin by gavage starting 3 days before FMT. Totally 16 times of FMT were performed throughout the 8-weeks experiment period. Feces were collected at week 8 for pyrosequencing. Intestinal tumor development and cell proliferation were determined. Inflammation of intestinal tissue and fecal fluid- incubated- colon tumor cell lines were detected by realtime-PCR. Immunofluorescence double staining was used to assess the activation of mononuclear-phagocyte system in tumor microenvironment after FMT. Tumor associated signaling pathway were examined by IHC and western blot. Part 3 To further determine the crucial role of gut microbiota in intestinal carcinogenesis, antibiotics were administrated to assess whether antibiotics can prevent DCA-induced intestinal tumor development.Results Part 1（1） No tumors were found and the intestinal tissue was normal in WT group. Small intestinal adenomas, which were identified as mostly tubular adenoma, sometimes accompanied with low-grade dysplasia in control group. In DCA group, the total number of intestinal adenomas were significantly increased, especially in middle and distal segment of small intestine and also in the colon. The adenomas in DCA group were distributed in intense and clusters, some large adenomas were also seen in colon. HE staining showed distal intestinal and 70% of colonic carcinogenesis in DCA group, remaining 30% of colorectal cancer as high grade of dysplasia. DCA promoted proliferation of intestinal tumor cells and inhibited apoptosis.（2） DCA decreased the diversity of intestinal microbiota and disrupted the balance of Firmicutes/ Bacteroidetes. DCA aggravated the gut dysbiosis, presented by increased abundance of pathogens and decreased abundance of probiotics.（3） DCA up-regulated the level of inflammatory cytokines in intestine.（4） DCA damaged the intestinal barrier, with decreased expression of proteins referred to intestinal tight junctions and also increased the intestinal permeability. Part 2（1） No tumors were found in FMT group 1. FMT group 3 had the largest adenoma burden and 72.7% of the adenomas in proximal section of small intestine were intramucosal cancer. The percentage of Ki-67 positive tumor cells was significantly increased in FMT group 3.（2）PCA analysis showed the gut microbiota community had significantly difference in dimensionality of PC2 and PC3 between FMT group 2 and FMT group 3. The ratio of Firmicutes and Bacteroidetes was decreased after colonization of fecal microbiota from DCA-treated mice. Pathogens such as Escherichia-Shigella, Bacteroides, Desulfovibrio and Helicobacter gasseri were increased, while probiotics such as Lactobacillus, Roseburia, Ruminococcus, Lachnospiraceae and Clostridium leptum were decreased significantly in FMT group 3.（3） After incubation with fecal contents from DCA group, colon tumor cell lines（IMCE and HT-29） had higher levels of inflammation cytokines. Besides, transplantation of fecal fluid from DCA group significantly induced intestinal inflammation to benefit the formation of tumor microenvironment, and also activated the polarization of macrophages.（4） Gut dysbiosis induced by DCA up-regulated the expression of TLR4 and My D88 and activated Wnt/β-catenin signaling pathway. Part 3 Manipulation of intestinal microbiota overally inhibited the progress of adenoma-adenocarcinoma sequence.Conclusions DCA-mediated dysbiosis can damage the intestinal barrier and induce the intestinal low-grade-inflammation, promotes the recruitment of tumor-associated macrophages and polarization of M2 macrophages, and further accelerates intestinal adenoma-adenocarcinoma sequence via activation of tumor-associated signalling pathway.