Multi-omics Investigation Of Host-microbiota Interaction In Colorectal Cancer And The Protective Role Of Bifidobacterium Bifidum

Backgrouds:Colorectal cancer（CRC）is the third most commonly diagnosed cancer and second leading cause of cancer death in both men and women globally,leading to an estimated551,269 annual deaths worldwide.The human intestine harbours a complex microbial community comprising more than 10¹⁴microorganisms;it carries greater than 150 times the number of genes in the human genome.In recent years,multiple studies support the critical role of the human gut microbiota in the carcinogenesis of CRC.The application of probiotics might become a promising treatment and prevention strategy for CRC.Bifidobacterium bifidum（B.bifidum）is a bacterial species belonging to the genus Bifidobacterium,which is one of the most widely used probiotic bacteria.However,there still lacks a comprehensive analysis of host-microbiota interactions using multi-omics datasets.So,the aim of this study is to further explore the role of gut microbiota in the carcinogenesis and prevention of CRC through high-throughput sequencing,multi-omics integration and system biology methods.Methods:The study includes two part.In the first part,we collected matched tumour and normal colon mucosa samples from thirty-six CRC patients.Using tumour-normal pairs from subsets in this sample collection,we generated microbiome and metabolome profiles,as well as host DNA methylation and transcriptome profiles.In the second part,we induced colorectal cancer in Male C57BL/6 mice by an intra-peritoneal injection of AOM（10 mg/kg）and three 7-day cycles of 2%DSS in drinking water with a 14-day recovery period between two consecutive DSS administrations.B.bifidum（3×10⁹CFU/m L）was gavaged once daily during the recovery period in one CRC mouse model group.Then,the faecal microbiome,faecal metabolome and colon mucosa transcriptome were profiled respectively.Results:Part 1:1. The study found that taxa in Halomonadaceae,Halomonas,Shewanella,genus Fusobacterium,Fretibacterium,Peptostreptococcus and Klebsiella were highly enriched in tumour colon mucosa（p-value<0.05）,while Paeniclostridium,Bacteroides eggerthii,Lactobacillus,Prevotella 2,Enterococcus,Clostridium perfringens and Veillonella were less abundant in tumour colon mucosa （p-value<0.05）.2.The metabolome analysis revealed that the levels of 4-hydroxybutyrate acid（4-HB）,lyxose,oxalic acid and D-talose were lower in tumour colon mucosa compared to the paired normal colon mucosa.On the other hand,the concentrations of lactic acid,glutamic acid,taurine and tetracosane were elevated in tumour colon mucosa.3.The transcriptome and DNA methylation analysis revealed sixteen DNA methylation related differentially expressed genes,with the expression of EPHB1,FREM2,GAD1,GRIN2D,PLCB1 higher in the tumour colon mucosa（gene promotor demethylated）and the expression of FCRLA,FOLR2,GNG2,GPNMB,CHP2,IGSF9,LSP1,MT1M,PI16,RELN and SCG2 lower in the tumour colonmucosa（gene promoter hypermethylated）.4.The correlations were identified between the change in abundance of microbial taxa,butyrate-related colonic metabolites,and methylation-associated host geneexpression in tumour colon mucosa compared with the adjacent normal colonmucosa.The increase of genus Fusobacterium abundance was significantlycorrelated with a decrease in the level of 4-HB and expression of immune-relatedpeptidase inhibitor 16（PI16）,Fc Receptor Like A（FCRLA）and LymphocyteSpecific Protein 1（LSP1）.The decrease in the abundance of another potentially4-HB-associated genus,Prevotella 2,was also found to be significantly correlatedwith the down-regulated expression of metallothionein 1M（MT1M）.Additionally,the increase of glutamic acid-related family Halomonadaceae was significantlycorrelated with the decreased expression of reelin（RELN）.The decreasedabundance of genus Paeniclostridium and genus Enterococcus were significantlycorrelated with increased lactic acid level,and were also linked to the expressionchange of Phospholipase C Beta 1（PLCB1）and Immunoglobulin SuperfamilyMember 9（IGSF9）respectively.Interestingly,4-HB,glutamic acid and lactic acidare all butyrate precursors,which modify gene expression by epigenetic regulationincluding DNA methylation.Part 2:1.Compared to PBS group and AOM.BF group,the AOM group had increased relativeabundance of Bacteroidetes,Parasutterella,Coriobacteriaceae,Actinobacteria,Peptococcaceae,Clostridiaceae,Ruminiclostridium＿9 and Streptococcaceae.2. The administration of B.bifidum attenuated tumourigenesis in the CRC mouse model.In addition,B.bifidum pre-treatment increased the relative abundance ofAkkermansia,Desulfovibrionaceae,Romboutsia,Turicibacter,Verrucomicrobiaceae,Ruminococcaceae＿UCG＿013,Lachnospiraceae＿UCG＿004and Lactobacillus.3. Meanwhile,B.bifidum altered metabolites involved in Retrograde endocannabinoid signaling,ABC transporters,Sphingolipid metabolism,Glycolysis/Gluconeogenesis,Glutathione metabolism,Glycerolipid metabolism,Galactose metabolism,Proteindigestion and absorption.4. The transcriptome analysis revealed genes differentially expressed between AOM group and PBS group as well as between AOM group and AOM.BF group.Thosegenes were involved in the regulation of toll-like receptor 4 signaling pathway,antimicrobial humoral immune response mediated by antimicrobial peptide,epidermal cell differentiation and regulation of epithelial cell proliferation.5. The relationship between the microbiota,metabolome and transcriptome were further assessed.The relative abundance of Romboutsia was significantly negativelycorrelated with the lactic acid level and significantly positively correlated with thecitric acid level.Furthermore,significant positive correlations with citric acidconcentration were also found with the relative abundance of Turicibacter,Lachnospiraceae＿UCG＿004.The relative abundance of Ruminiclostridium＿9 was,however,significantly negatively correlated with the citric acid level.Additionally,Peptococcaceae and Ruminiclostridium＿9 exhibited a significant negativerelationship with cadaverine.The differentially expressed genes were significantlyassociated with at least one of the differentially abundant metabolites and thedifferentially abundant microbes.Conclusions:1.Our study identified significant associations between previously reportedCRC-related microbial taxa,butyrate-related metabolome and DNAmethylation-associated gene expression in tumour and normal colon mucosa fromCRC patients,which uncovered a possible mechanism of the involvement ofmicrobiota in the carcinogenesis of CRC.In addition,these findings offer insightinto potential new biomarkers,therapeutic and prevention strategies for CRC.2.These findings supported the beneficial role of B.bifidum in intestinal health bymodulating dysbiosis and the gut metabolic profile as well as potentially influencingfunctional gene expression.The manipulation of the gut microbial compositionusing probiotics might be a promising prevention strategy for CRC.Long-term andlarge-scale clinical trials are warranted for the potential clinical applications of thisstrategy in the future.