Gut Microbiota From Patients With Colorectal Cancer Enhances The Progression Of Intestinal Adenoma In Mice By Activating The Wnt Signaling Pathway

ObjectiveColorectal cancer（CRC）,ranks the third in morbidity and mortality,is one of the most common malignant tumors in the world.The incidence and prevalence of colorectal cancer are increasing especially in China.Current studies suggest that CRC is a disease caused by multiple factors and multiple steps.The currently known risk factors of CRC including genetic predisposition and environmental factors such as age,gender,obesity,lifestyle.About 95%of intestinal tumors develop from adenomas,however its mechanisms have not been fully illuminated.Recent studies have found that the composition and function of intestinal microbiota in CRC patients are significantly different from that in healthy people.And pathogenic bacteria are more likely to colonize the intestinal cavity.The interaction between direct carcinogenicity of intestinal microbiota and its derived factors activates oncogenic signaling pathways.To some extent,CRC seems to be considered a bacteria-related disease.This study aimed to explore the role and mechanism of disordered intestinal microbiota in the progression of intestinal adenoma to adenocarcinoma.Methods1.Human experimental method:We collected fresh feces from 10 healthy subjects and 10 CRC patients to prepare faecal suspension according to standard procedures.All subjects who were screened by strict inclusion and exclusion criteria signed the informed consent.The faecal suspension was stored separately in-80°C refrigerator for subsequent experiments.2.Animal experimental method:After receiving three days of antibiotic cocktail pretreatment,the twenty conventional female C57BL/6J mice aged four weeks were randomly divided into two groups,one group was gavaged fecal samples from healthy controls,while the other group was gavaged fecal samples from CRC patients.The thirty Apc^min/+female mice aged 4 weeks were randomly divided into three groups,which were gavaged sterile PBS,fecal samples from healthy controls and fecal samples from CRC patients,respectively.The fecal microbiota transplantation（FMT）was repeated 16 times during the experimental period of 8 weeks.Fresh faeces from each mouse were collected before sacrifice.Then the liver,spleen,small intestine and colon tissues were isolated.The tissue of the small intestine was divided into three parts on average.The tissue near the stomach was defined as the proximal segment,and the tissue near the ileocecum was defined as the distal segment.The proximal tissue of each intestinal segment was snap frozen in liquid nitrogen and kept at-80°C later,and the distal tissue was fixed with 4%paraformaldehyde.（1）The number and distribution of intestinal adenomas of mice in each group were recorded.HE staining was performed on paraffin-embedded intestinal adenoma tissue to evaluate the pathological types.（2）Ki-67 immunohistochemical staining was performed on paraffin sections of intestinal adenomas in mice to evaluate the proliferation of intestinal tumor cells.Terminal deoxynucleotidyl transferase d UTP nick end labelling（TUNEL）was used to detect tumour cellular apoptosis.（3）Real time-PCR was performed to detect the intestinal barrier function and the expression of inflammatory factors.（4）Western blot was used to detect the expression of proteins related to the Wnt signaling pathway in intestinal tumor tissues.（5）16S r DNA Amplicon sequencing was used to detect the gut microbiota of mice under different treatment conditions.Results（1）The total number of adenomas in mice receiving fecal samples from CRC patients was increased compared with the PBS group,and the mice receiving fecal samples from healthy controls.（2）Compared with the mice receiving fecal samples from healthy controls,the positive percentage of Ki-67 immunohistochemical staining was significantly increased and the percentage of apoptotic cells was significantly decreased in the adenoma cells of mice receiving fecal samples from CRC patients.（3）The relative m RNA expression of tight junction protein associated with intestinal barrier function were decreased and the relative m RNA expression of intestinal inflammatory cytokines were increased in mice receiving fecal samples from CRC patients compared with mice receiving fecal samples from healthy controls.（4）Compared with mice receiving fecal samples from healthy controls,the mice receiving fecal samples from CRC patients showed significant heterotopic expression ofβ-catenin in intestinal adenoma cells,and the proportion of cyclin D1 positive cells increased significantly.（5）Compared with the mice which gavaged by the fecal samples from healthy controls,the abundance of Prevotella and Helicobacter increased in the mice which gavaged by the fecal samples from CRC patients.Meanwhile,the abundance of short-chain fatty acids（SCFAs）producing bacteria,such as ClostridiumⅣ,Clostridium XIVa,and Roseburia were significantly lower.ConclusionThe Apc^min/+mice which gavaged by the fecal samples from CRC patients showed intestinal barrier disruption,chronic low grade inflammation,activation of Wnt/β-catenin signaling pathways,and imbalance gut mcrobiota to promote the progression of intestinal adenoma.This study provided a new perspective for the prevention of CRC against gut mcrobiota.Targeted intervention against gut mcrobiota seems to be a promising way to treat CRC.