Table 1.
Clinical characteristics of CRC patients with or without F. nucleatum infection.
Fig 1.
MiR-4474 and miR-4717, which were screened and identified as being associated with F. nucleatum-induced CRC.
(A and B) Schematic flow of the study. Fn = Fusobacterium nucleatum; control = paracancerous tissues; CRC = colorectal cancer. Total RNA from CRC and adjacent normal tissues with or without F. nucleatum infection were used to perform the microarray assay. (C) The down- and upregulated miRNAs in hierarchical clustering analysis. Two subclasses, F. nucleatum-positive CRC (n = 10) and F. nucleatum positive control (n = 5), exhibited clustering results of 49 miRNAs with 2-fold changes in F. nucleatum-induced CRC.
Fig 2.
CREBBP is the primary aberrantly expressed gene in F. nucleatum-induced CRC.
(A and B) Hierarchical clustering analysis of aberrantly expressed genes in F. nucleatum-induced CRC. Up- and downregulated genes are shown in red and green, respectively. The fold changes in mRNA expression in F. nucleatum-positive CRC versus the F. nucleatum positive control. (C) GO enrichment analysis of aberrantly expressed mRNAs. The vertical axis represents the pathway category, and the horizontal axis represents the enrichment of pathways. The most enriched GO pathways were screened in accordance with P<0.001 and FDR<0.05. (D) KEGG pathway analysis of aberrantly expressed mRNAs. The most enriched pathways were filtered by the criteria of P< 0.001 and FDR < 0.05. (E) The protein-protein interaction network of the major aberrantly expressed genes was generated. Up- and downregulated genes are shown in red and blue, respectively.
Fig 3.
Expression of miR-4474/4717 and CREBBP mRNA by real-time RT-PCR analysis.
(A and B) The expression of miR-4474/4717 was detected by real-time RT-PCR in 15 CRC patients and 10 paracancerous tissues with or without F. nucleatum infection. (C and D) The expression of CREBBP mRNA was detected by real-time RT-PCR in clinical specimens. (E and F) The expression of CREBBP mRNA was detected by real-time RT-PCR in Caco-2 cells after F. nucleatum infection 24 hours. The results shown are representative of at least three independent experiments. *P<0.05.
Fig 4.
CREBBP is a novel target of miR-4474/4717.
(A) The sequences of miR-4474/4717 and the potential binding site in the 3’UTR of CREBBP. (B and E) Luciferase reporter assay. HEK293 cells were transiently cotransfected with 0.8 μg of pMIR-CREBBP-wt or pMIR-CREBBP-mut, 0.04 μg of Renilla luciferase control vector, and 100 nM miR-4474/miR-4717 control, mimic, or inhibitor with Lipofectamine 2000 for 24 h. (C,F, D and G) Western blot and qRT-PCR analyses. Human epithelial colorectal Caco-2 cells were transfected with miR-4474/4717 control, mimic or inhibitor for 24 h, and the mRNA and protein expression of CREBBP was detected by qRT-PCR and Western blot analyses. The results shown are representative of at least three independent experiments. *P< 0.05.