The Role Of Intestinal Microbiota In The Pathogenesis Of Colorectal Cancer And Its Diagnostic Value: Based On Multi-omics

Colorectal cancer(CRC)is one of the most common digestive tract tumors.Its morbidity and mortality are increasing in our country,which may be closely related to the westernization of diet habits,the increase of red meat intake,the decrease of dietary fiber intake and so on.The occurrence and progression of CRC is the result of the interaction of host gene sensitivity,environmental and dietary factors.Intestinal dysbacteriosis caused by diet is an important cause of CRC.With the development of high-throughput sequencing technology,intestinal microbiota have been confirmed to play a key role in the pathogenesis of colorectal cancer.At present,it has been reported that the transplantation of feces from patients with colorectal cancer into mice can induce epithelial cells with the pathological progress of "normal colonic epithelium-intraepithelial neoplasia-colorectal cancer" in mice,preliminary establishing the causal relationship between intestinal microbiota and CRC.However,whether the direct toxicity of specific strains,or intestinal microbiota affecting the immune environment,or the metabolites of intestinal microbiota causing the tumor is is still obscure,not to mention their clear molecular mechanisms.It’s exciting that multi-omics research has developed a new idea and method for studying the pathogenic effect of intestinal microbiota and it is also a hot spot in tumor research at present.Through the combination of genomics,metabolomics,microbiome.,and immunology and other multi-group overall,cross-study can contribute to deeply understanding the role of intestinal microbiota in the pathogenesis of CRC,even achieving clinical transformation.Although the multi-omics study of intestinal microbiota has made important progress in metabolic diseases such as diabetes and obesity,it is still in the exploratory stage for CRC reasearch.Therefore,,this study will combine microbiome,metabolomics and immunology to explore the impact of intestinal microbiota on the occurrence and development of CRC and find the clinical value of combined microtiota in CRC diagnosis.Part 1 Study on the correlation between fecal microbiome and blood metabolomics in colorectal cancer patients[Objective]The aim of the first part of this study was to explore the different intestinal micribiota abundance in colorectal cancer patients with adenomatous polyps and normal people.The plasma metabolomics and bile acid of the matched population were detected,and the correlation analysis between microbiome and metabolomics was conducted to study the possible mechanism of intestinal microbiota causing colorectal cancer from the perspective of metabolomics.[Methods and meterials]Select 60 healthy people,50 adenoma patients and 130 CRC patients confirmed by endoscopy and histopathology in ChangZheng hospital from January to May 2018 and collect their stool and plasma samples.16sRNA V4 variable region were pyrosequenced and the analyses of diversity index,PCA,LefSe were adopted to compare the species and diversity of microbiota in each group.Distinguish the diversity and structure of intestinal microbiota in CRC,adenoma patients and normal subjects.Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry(UPLC-ESI-QTOF-MS)was performed to characterize the metabolic profile of plasma.Bile acids in Plasma were detected by liquid Phase Mass Spectrometry.The software of data acquisition and processing is MassLynx Acquisition and Progenesis QI,and the statistical analysis software of,is EZinfo3.0.3.0.A pairwise comparison of CRC group,adenoma group and normal group showed differences in plasma metabolites and bile acids.The Spearman correlation between microbiome species,KEGG pathway and metabolomics was analyzed by R 3.50 software package.[Results]In CRC group,clostridium and streptococcus abundance were significantly increased.The fusobacterium was highly relative to the onset of"normal-adenoma-CRC" model.The plasma bile acid in colorectal cancer group,polyp group and normal group was significantly different by mass spectrometry.Primary bile acids:cholic acid(CA),goose deoxycholic acid(CDCA),ursodeoxycholic acid(UDCA)and primary bile acid combined with glycine(GCA,GCDCA,GUDCA)were significantly reduced in the colorectal cancer group(p<0.05).Secondary bile acids:licholic acid(LCA),deoxycholic acid(DCA)and secondary bile acid combined with glycine(GLCA,GDCA)were significantly reduced in the colorectal cancer group(P<0.05).But bile acid combined with taurine were no significant difference.Mass spectrometric analysis of plasma small molecule metabolomics showed that there were 180 metabolites significant differently between the colorectal cancer group and the normal group.There are eight metabolites significant differences in normal-adenoma-tumorigenesis progress.Finally,metabolomics and 16srDNA microbiome association analysis revealed that clostridium abundance and histidine metabolism significantly increased in the tumor group(P<0.05),which resulted in the rise of 6 tumor-related metabolites,including 12a.Hydroxy3-oxygen bile acid and phosphocholine.[Conclusion]Clostridium plays an important role in the progression of"normal epithelium-adenoma-carcinogenesis’,.Clostridium mediates the occurrence of colorectal cancer through histidine metabolism,which leads to elevated concentrations of tumor-related metabolites in the blood such as 12a.Hydroxy3-oxygen bile acid and phosphocholine.This may be one of the important pathogenesis of colorectal cancer.Part 2 Intestinal microbiota promote the occurrence and development of colorectal cancer by mediating inflammatory release and immune microenvironment disorder[Objective]The aim of the second part was to study the effects of intestinal microbiota on mucosal barrier,local and systemic inflammatory response,and tumor immune microenvironment in the process of colorectal cancer,and to explore intestinal microbiota in the pathogenesis of colorectal cancer from the view of immunology.[Materials and methods]Select 20 healthy people,20 polyp patients and 26 CRC patients confirmed by endoscopy and histopathology in ChangZheng hospital from March to May 2018 and collect their plasma and Intestinal tissue samples:Normal intestinal tissue,polyp adenoma tissue,tumor tissue(T),paracancerous tissue(TP)and normal tissue(N).The distribution and structure of tissue microbiota were analyzed by 16srDNA sequencing.The distribution of Fusobacterium nucleatum(Fn)and Escherichia coli(E.coli)in colorectal cancer tissues and metastatic lymph nodes were studied by immunofluorescence in situ hybridization(FISH).The expression of intestinal barrier-associated proteins MUC2,ZO-1,Occludin,claudinl and the expression inflammatory immune-related proteins MyD88,IDO-1,p-STAT3,ATF6 and IRAK-M,PD-Ll were detected by immunofluorescence and western blot.Immunohistochemistry was used to detect the expression of Ki67.Biochemical method analyzes plasma lactic acid,ELISA analysis common inflammatory factors in tissue and blood.The number of CD4+T,CD8+T,TREG,DC and B cells in blood were analyzed by flow cytometry,and the abundance of Fn in tissues was detected by Real-time PCR.[Results]There was no significant difference in the diversity of microbiota between tumor tissues and normal tissues,but the abundance of Clostridium increased and the abundance of Gemmiger decreased in tumor tissues.FISH found that Fn and E.coli increased significantly in tumor tissues and appeared in metastatic lymph nodes.The plasma lactic acid in CRC group was significantly higher than that in control group.Compared with polyps and normal intestinal tissues,western blot and immunofluorescence showed that the expression of ZO-1 and claudin-1 mucosal barrier-associated proteins in tumor tissues was significantly lower(p<0.05).The expression inflammation-related proteins Myd88,IDO-1 and p-STAT3 was significantly increased(p<0 05),and the expression of anti-bacterial protein IRAK-M was significantly lower(p<0 05)in CRC.The expression of PD-L1 protein was significantly increased(p<0 05)in CRC.In patients with colorectal cancer,compared with normal tissues,western blot and immunofluorescence in tumor tissue revealed that claudin 1 mucosal was significantly reduced(p<0.05),Myd88,IDO-1,p-STAT3 was significantly increased(p<0.05),and the expression of antimicrobial protein IRAK-M was significantly reduced(p<0.05).The levels of inflammatory cytokines IL-10,TNFa.,IL-6,IL-1β,TGF-β,IFN-Υ,IL-17,and IL-23,CRP in the plasma of the tumor group a were significantly increased by ELISA(p<0.05)than normal group,while IL-12 deceased significantly(p<0.05).Compared with polyps and normal intestinal tissues,the contents of inflammatory cytokines such as IL-10.,TNFα,IL-6,IL-1β,TGF-β,IFN-Υ,IL-17 and IL-23 were also significantly increased in tumor tissues by ELISA(p<0.05),while IL-12 was significantly decreased(p<0.05).tumor issue and lymph node compared with normal tissues,the contents of inflammatory cytokines IL-10,TNFα,IL-6,IL-1β,TGF-β,IFN-Υ,IL-17 and IL-23 were significantly increased by ELISA(p<0.05)and while IL-12 was significantly decreased(p<0.05).Flow cytometry revealed that NK cells were significantly reduced in the tumor group compared with the normal group.qPCR showed that compared with normal intestinal tissues,the abundance of Fn in tumor tissues and metastatic lymph nodes was significantly increased than normal tissue(p<0.05),which was closely related to inflammatory factors and Ki67.[Conclusion]CRC tissue is characterised of disfunctional intenstinal barrier and disorder microbiota.Pathogenic bacteria(Fn bacteria)directly contact with intestinal immune cells,mediating local and systemic inflammatory response,and inhibiting the activation of NK cells.It results in the imbalance of immune microenvironment and promotes the occurrence and development of colorectal cancer.Part 3 The diagnostic value of combined microbiota detection in colorectal cancer[Objective]The aim of the third part was to screen the differences in fecal and blood abundance of pathogenic bacteria(Fn,ETBF and E.coli)and beneficial bacteria(Fp,Lb and Bb)in normal healthy people,colorectal cancer patients,adenomatoid polyps patients and explore the non-invasive diagnostic value of combined bacteria detection in colorectal cancer.[Materials and methods]Select 80 healthy people,160 polyp patients and 240 CRC patients confirmed by endoscopy and histopathology in ChangZheng hospital from January 2017 to October 2018.The adenoma group was divided into progressive adenoma group and non-progressive adenoma group.According to TNM staging,stage I and stage II colorectal cancer was defined as early colorectal cancer,and stage III and IV were defined as late colorectal cancer.Stool,blood and tissues samples from case and health group were collected respectively according to strict sampling standards.qPCR was used to detect the differences in the abundance of bacteria in stool,blood and tissues.The differential flora was selected,the ROC curve was drawn and the test efficacy in the diagnosis of colorectal cancer was calculated.by different combinations of the differential flora.[Results]Compared with normal and adenoma group,Fn bacteria abundance in stool,blood and tissue increased significantly in CRC group(p<0.05).Fecal and tissue ETBF bacteria abundance increased significantly(p<0.05),but blood ETBF abundance between groups have no significant difference.There was no significant difference in the abundance of E.coli bacteria in stool and blood,but the abundance of E.coli in early colorectal cancer group was significantly higher than normal group(p<0.05),IT was no significant difference in Bb abundance in stool and blood.However,the abundance of Bb in advanced adenomas was significantly lower than that in non-advanced adenomas(P<0.05).the abundance of Lb in stool and tissues was significantly lower than that in non-advanced adenomas(p<0.05),but there was no significant difference in blood Lb content among the three groups.The abundance of Lb in stool and tissues of tumor group decreased significantly(p<0.05),but there was no significant difference in blood Lb abundace among the three groups There was no significant difference in Fp bacteria in stool,blood and tissue among the three groups(p>0.05).The abundance of fecal Fn+ETBF bacteria and blood Fn bacteria as diagnostic tools of colorectal cancer(AUC=0.763)is better than that of stool Fn bacteria(AUC=0.689)and combined fecal Fn+ETBF(AUC=0.731)in the diagnosis of colorectal cancer.[Conclusions]The stool of patients with colorectal cancer are rich in Fn bacteria and ETBF bacteria,and the abundance of Fn bactera in the blood is higher than that of normal healthy people and patients with colorectal adenomatoid polyps.Fecal Fn bacteria and ETBF bacteria combined with blood Fn bacteria have the highest detection efficiency in diagnosis of colorectal cancer,which is an ideal noninvasive diagnostic value for colorectal cancer,but needs to be further verified by multi-center clinical trials.