©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
Relationship between Fusobacterium nucleatum, inflammatory mediators and microRNAs in colorectal carcinogenesis
Marcela Alcântara Proença, Joice Matos Biselli, Maysa Succi, Ana Elizabete Silva, Department of Biology, UNESP, Univ. Estadual Paulista, Campus of São José do Rio Preto, São José do Rio Preto, São Paulo 15054-000, Brazil
Fábio Eduardo Severino, Department of Surgery and Orthopedics, Faculty of Medicine, UNESP, Univ. Estadual Paulista, Campus of Botucatu, Botucatu, São Paulo 18618-687, Brazil
Gustavo Noriz Berardinelli, Rui Manuel Reis, Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo 14784-400, Brazil
Alaor Caetano, Endoscopy Center of Rio Preto, São José do Rio Preto, São Paulo 15015-700, Brazil
Rui Manuel Reis, Life and Health Sciences Research Institute, University of Minho, Campus Gualtar, Braga 4710-057, Portugal
Rui Manuel Reis, ICVS/3B’s-PT Government Associate Laboratory, Campus Gualtar, Braga 4710-057, Portugal
David J Hughes, Cancer Biology and Therapeutics Group, UCD Conway Institute, University College Dublin, Dublin D04 V1W8, Ireland
Author contributions: Proença MA performed the research and experiments, collected the data, performed statistical analyses, and wrote the paper; Biselli JM performed statistical analyses and quantification of Fusobacterium nucleatum; Severino FE constructed the microRNA:mRNA interaction network; Caetano A and Succi M collected the samples; Berardinelli GN and Reis RM performed the KRAS mutation and MSI status experiments; Hughes DJ designed the research experiments and reviewed the paper; Silva AE designed the research experiments, wrote the paper, and reviewed the paper; all authors read and approved the final manuscript.
Supported by São Paulo Research Foundation (FAPESP), No. 2012/15036-8; and National Council for Scientific and Technological Development (CNPq), No. 474.776/2013-1.
Institutional review board statement: This work was approved by the Ethics in Research Committee of CEP/IBILCE/UNESP, No. 1.452.373.
Conflict-of-interest statement: No conflict-of-interest.
Data sharing statement: Participants gave written informed consent for data sharing.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author to: Ana Elizabete Silva, PhD, Adjunct Professor, Teacher, Department of Biology, UNESP São Paulo State University, Rua Cristóvão Colombo 2265, São José do Rio Preto, São Paulo 15054-000, Brazil. firstname.lastname@example.org
Telephone: +55-17-322122384 Fax: +55-17-322212390
Received: October 30, 2018
Peer-review started: October 30, 2018
First decision: November 6, 2018
Revised: November 29, 2018
Accepted: December 13, 2018
Article in press: December 13, 2018
Published online: December 21, 2018
To examine the effect of Fusobacterium nucleatum (F. nucleatum) on the microenvironment of colonic neoplasms and the expression of inflammatory mediators and microRNAs (miRNAs).
Levels of F. nucleatum DNA, cytokine gene mRNA (TLR2, TLR4, NFKB1, TNF, IL1B, IL6 and IL8), and potentially interacting miRNAs (miR-21-3p, miR-22-3p, miR-28-5p, miR-34a-5p, miR-135b-5p) were measured by quantitative polymerase chain reaction (qPCR) TaqMan® assays in DNA and/or RNA extracted from the disease and adjacent normal fresh tissues of 27 colorectal adenoma (CRA) and 43 colorectal cancer (CRC) patients. KRAS mutations were detected by direct sequencing and microsatellite instability (MSI) status by multiplex PCR. Cytoscape v3.1.1 was used to construct the postulated miRNA:mRNA interaction network.
Overabundance of F. nucleatum in neoplastic tissue compared to matched normal tissue was detected in CRA (51.8%) and more markedly in CRC (72.1%). We observed significantly greater expression of TLR4, IL1B, IL8, and miR-135b in CRA lesions and TLR2, IL1B, IL6, IL8, miR-34a and miR-135b in CRC tumours compared to their respective normal tissues. Only two transcripts for miR-22 and miR-28 were exclusively downregulated in CRC tumour samples. The mRNA expression of IL1B, IL6, IL8 and miR-22 was positively correlated with F. nucleatum quantification in CRC tumours. The mRNA expression of miR-135b and TNF was inversely correlated. The miRNA:mRNA interaction network suggested that the upregulation of miR-34a in CRC proceeds via a TLR2/TLR4-dependent response to F. nucleatum. Finally, KRAS mutations were more frequently observed in CRC samples infected with F. nucleatum and were associated with greater expression of miR-21 in CRA, while IL8 was upregulated in MSI-high CRC.
Our findings indicate that F. nucleatum is a risk factor for CRC by increasing the expression of inflammatory mediators through a possible miRNA-mediated activation of TLR2/TLR4.
Core tip: We examined the influence of Fusobacterium nucleatum (F. nucleatum) in colorectal adenoma (CRA) and colorectal cancer (CRC) on the mRNA expression of inflammatory mediators and the association with microRNA (miRNA) levels, KRAS mutation, and microsatellite instability (MSI). We suggest that F. nucleatum contributes to CRC development by increasing the expression of inflammatory mediators through a possible miRNA-mediated activation of TLR2/TLR4. The miRNA:mRNA interaction network suggests an upregulation of miR-34a in CRC via a TLR2/TLR4-dependent response to F. nucleatum. KRAS mutations were more frequent in F. nucleatum-infected CRC and were associated with a greater expression of miR-21 in CRA, while IL8 was upregulated in MSI-high CRC.