Abstract: BACKGROUND: As an anaerobic bacterium in the gut, the regulatory mechanism of Fusobacterium nucleatum on miRNA expression in colonic mucosal cells remains unclear. Recent studies have highlighted the association between Fusobacterium nucleatum and colonic polyps, necessitating further investigation into regulatory mechanisms of Fusobacterium nucleatum on miRNA to elucidate polyp pathogenesis.
OBJECTIVE: To investigate the regulatory effects of Fusobacterium nucleatum on gene expression in colonic mucosal cells through miRNA-mediated microbe-host interactions and to elucidate its molecular mechanisms in promoting polyp formation through specific signaling pathways in mice.
METHODS: (1) Ten 6-8-week-old male C57BL/6 mice were randomly divided into control and infection groups. The mice in the control group received sterile PBS via gastric gavage, and those in the infection group were administered with Fusobacterium nucleatum suspension (10 μL/g), twice weekly for 3 months to establish a colonic polyp model. Fecal samples (7-8 pellets per group) were collected on days 30, 60 and 90. On day 91, the colonic tissues (from the anus to the ileocecal region) were harvested for colon length measurement and hematoxylin-eosin staining. Total RNA was extracted from colonic tissues of each group for quantitative analysis, library construction, sequencing, and bioinformatic processing, including miRNA clustering, differential expression analysis, and functional enrichment of Predict target genes (Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis). (2) The colonic mucosal epithelial cells of mice were collected and infected with Fusobacterium nucleatum at different multiplicity of infection values (0, 10, 50, 100, and 200) for 6 hours, and with Fusobacterium nucleatum at the multiplicity of infection of 200 for 6, 12, 24, and 48 hours, respectively. Cell viability was assessed by cell counting kit-8 assay. Additionally, a cell scratch assay was performed to evaluate cell migration ability after Fusobacterium nucleatum infection at the multiplicity of infection of 200 for 24 and 48 hours.
RESULTS AND CONCLUSION: (1) Intragastrical administration of Fusobacterium nucleatum for 12 weeks significantly inhibited body mass, shortened colon length, and induced mucosal gland atrophy with inflammatory cell infiltration in mice. qPCR confirmed a time-dependent increase in Fusobacterium nucleatum 16S rRNA gene copies in the infection group. (2) In vitro experiments demonstrated that Fusobacterium nucleatum solution with high multiplicity of infection values significantly enhanced colon epithelial cell proliferation (P < 0.05), and the absorbance values progressively increased over time. Cell scratch assays revealed enhanced cell migration in the infection group. (3) miRNA profiling identified 19 differentially expressed miRNAs, with principal component analysis showing distinct intergroup separation (PC1=61%). Clustering analysis revealed the downregulation of tumor-associated miRNAs (e.g., miR-143-3p and miR-145-5p). Target genes were enriched in “regulation of cell proliferation” (GO:0042127) and “pathways in cancer” (mmu05200). By integrating animal models, miRNA sequencing, and functional analyses, this study suggests that Fusobacterium nucleatum drives colonic mucosal hyperplasia and polyp formation by modulating tumor-suppressive miRNAs (e.g., miR-143-3p and miR-145-5p) to activate oncogenic signaling pathways. 

Key words: Fusobacterium nucleatum, miRNA, colonic polyps, microbial-host interaction, epigenetic regulation, exosomes, tumor microenvironment