Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Adenoma-adenocarcinoma Sequence In Apc^min/+ Mice

Objective:human body and microorganism are in symbiosis.In the role of pathogenic factors,intestinal microorganisms will appear imbalanced,resulting in flora disorder,increase of pathogenic bacteria,decrease of beneficial bacteria,thus inducing intestinal inflammation and stimulating tumor growth.At present,most of the related researches focus on the detection and correlation analysis of intestinal flora in patients with intestinal tumor.What is the mechanism of intestinal flora imbalance in the progression and transformation of adenoma?What are the key factors involved in this process?What are the key signaling pathways?The purpose of this paper is to discuss these problems.Methods:Patients with colorectal cancer diagnosed by pathology were selected from endoscopy center and gastroenterology department of General Hospital of Tianjin Medical University.At the same time,10 healthy people were selected to collect the fresh feces.The collected fresh feces were put into the feces box,marked and stored in-80℃refrigerator.C57BL/6J mice of SPF grade and Apc^min/+mice of SPF grade were selected.They were divided into four groups:FMT-CH group（C57BL/6J mice transplanted with fecal bacteria solution of healthy subjects）,FMT-CC group（C57BL/6J mice implanted with fecal bacteria solution of patients with colorectal cancer）,FMT-AH group(Apc^min/+mice implanted with fecal bacteria solution of healthy subjects)and FMT-AC group(Apc^min/+mice implanted with fecal bacteria solution of patients with colorectal cancer).In order to facilitate the colonization of bacteria in the intestinal tract,mice in each group were given drinking water containing 0.2g/l ampicillin+0.2g/l neomycin+0.2g/l metronidazole+0.1g/l vancomycin three days before the experiment.The first week of fecal bacteria transplantation（FMT）,once a day,7 times in total;the second week of FMT,once every other day,3 times in total;the third week of FMT,once a week,6 times in total;the whole experiment process,a total of fmt16 times.During the whole FMT process,the basic condition of mice should be observed every day,including activity state,water consumption and blood trace in the cage,and the weight should be weighed once a week.Before and after the experiment（the 9th week）,fresh feces of mice were collected for 16S r RNA pyrosequencing.q PCR was used to detect the levels of various inflammatory cytokines and colon mucosal barrier related factors（including ZO-1,claudin3,occludin,MUC2,cryptdin,NLRP3,IL-1βand TNF-α）.Immunohistochemical Ki-67 staining.Western blot was used to detect the expression of totalβ-Catenin,Cyclin D1,c-myc andβ-actin.The total RNA of colonic mucosa was separated,the RNA quality was measured by spectrophotometer,and reverse transcription was carried out with c DNA conversion kit.The gene expression in colon of Apc^min/+mice was detected by RT-PCR.Calculate the fold change of sample quality inspection expression quantity,select the differential expression gene>2 or<0.5,and discuss the mechanism of differential expression gene to adenoma canceration and clinical prognosis analysis.ResultsThe first part（1）The selection of fecal fluid donors for colorectal cancer met the inclusion criteria.There was no significant difference in the mean age of fecal fluid donors between colorectal cancer and healthy subjects（61.9±6.72 vs.65.70±9.91）.There was no significant difference in BMI between the two groups（21.39±1.67 vs.20.84±1.60）.（2）The relative abundance of Roseburia in fecal mixed supernatant of CRC patients was decreased at generic level compared with that of healthy people.The relative abundance of akkermansia in fecal mixed supernatant of CRC patients was higher than that of healthy people.The second part（1）At the end of 8th week,there was no adenoma in the whole intestine of FMT-CC group and FMT-CH group.In the FMT-AH group,most of the intestinal adenomas were scattered in the proximal and middle part of the small intestine,while in the FMT-AC group,the total intestinal adenomas were significantly increased,and some of the proximal colon adenomas were significantly increased.Pathological results showed that 30%of the intestinal adenomas in FMT-AH group showed high-grade intraepithelial neoplasia,90%of the intestinal adenomas in FMT-AC group showed high-grade intraepithelial neoplasia,and some of the adenomas showed intramucosal carcinoma.（2）The percentage of Ki-67 positive cells in intestinal tumor of FMT-AC group was significantly higher than that of FMT-AH group.Western blot was used to detect the protein levels of totalβ-Catenin,Cyclin D1 and c-myc in small intestinal tumors.The protein levels of FMT-AC group were significantly higher than that of FMT-AH group.（3）The relative m RNA expression of ZO-1,claudin3,oclaidin,MUC2 and cryptdin in Apc^min/+mice in FMT-AC group was significantly lower than that in FMT-AH group.（4）The expression of IL-1β,NLRP3 and TNF-αin intestinal epithelial cells of FMT-AC group was significantly higher than that of FMT-AH group.（5）The level of short chain fatty acids（including acetic acid,propionic acid and butyric acid）in the ileocecal region of Apc^min/+mice in FMT-AC group was significantly lower than that in FMT-AH group.（6）After FMT transplantation,the feces of FMT-AH group and FMT-AC group were sequenced with 16S r RNA.At the genus level,the results showed that the pathogenic bacteria prevotella in the intestinal flora of FMT-AC group were significantly higher than that of FMT-AH group,and the difference was statistically significant.However,probiotics parasutterella decreased compared with FMT-AH,but there was no significant difference.The third part（1）The differentially expressed genes between fmt-ac and fmt-ah were screened by high-throughput RNA sequencing.278 differentially expressed genes were obtained,including 228 up-regulated genes and 50 down-regulated genes.（2）The function of the differential gene（DEG）was annotated.GO analysis showed that Wnt protein binding,lipid metabolism and immune response pathway were significantly enhanced.（3）KEGG analysis showed that the differentially expressed genes from annotation to KEGG were mainly distributed in Ras signaling pathway,Wnt signaling pathway,NF-κB signaling pathway and T cell receptor signaling pathways.（4）The network interaction analysis of the differentially expressed genes of hubba gene was carried out by using the Cytoscape tool,and hubba genes of interest was Esr1.Using TCGA database,there was differential expression in colon cancer and adjacent tissues,and further survival analysis and immune infiltration analysis were carried out.（5）In terms of overall survival rate,different expression levels of ESR1 directly affect the survival time of the body and have a significant relationship with the total survival time of patients.（6）There was a significant correlation between the expression of ESR1 gene and B cells,CD8+T cells and macrophages.Conclusions:1.The intestinal flora of colorectal cancer patients can promote the canceration of colorectal adenoma as a whole.2.The disturbance of intestinal flora may promote the carcinogenesis of adenoma by promoting inflammatory response,reducing the concentration of short chain fatty acids and activating Wnt signaling pathway.3.RNA-seq results showed that Wnt protein binding,lipid metabolism and immune response pathway were significantly enhanced,which may be closely related to adenoma carcinogenesis.4.ESR1,a differentially expressed gene,participates in immune escape and promotes the development of colorectal adenoma.