Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 21, 2018; 24(47): 5351-5365
Published online Dec 21, 2018. doi: 10.3748/wjg.v24.i47.5351
Relationship between Fusobacterium nucleatum, inflammatory mediators and microRNAs in colorectal carcinogenesis
Marcela Alcântara Proença, Joice Matos Biselli, Maysa Succi, Fábio Eduardo Severino, Gustavo Noriz Berardinelli, Alaor Caetano, Rui Manuel Reis, David J Hughes, Ana Elizabete Silva
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:
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.
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
Research background

Recently, Fusobacterium nucleatum (F. nucleatum), an anaerobic bacterial component of the oral and gut commensal flora, has emerged as a risk factor for colorectal cancer (CRC) development. Several studies have observed an association between overabundance of F. nucleatum in colonic tumor tissue compared to the normal matched mucosa. However, despite progress in this field the molecular mechanisms of how the bacterium etiologically contributes to carcinogenesis are still unclear.

Research motivation

We previously observed an association of the TLR2-196 to -174del genetic variant with increased CRC risk, together with an increased expression of TLR2 mRNA and protein in tumor tissues[31]. The major postulated mechanism of F. nucleatum-mediated colorectal tumorigenesis involves immune related inflammatory responses. Therefore, we decided to extend our previous work by measuring the transcript levels of important mediators in the pathogen-activated immune and inflammatory response, including TLR2 / TLR4 receptor and cytokine genes, and then evaluating the association of their expression with F. nucleatum levels in colorectal tumors. As microRNAs have been shown to be epigenetic regulators of inflammatory responses, we further examined the involvement of miRNAs in modulating the bacterial - cytokine interaction.

Research objectives

The main objective of this study was to investigate the association between inflammatory genes and F. nucleatum in colorectal carcinogenesis, by examining tissues from the major colorectal neoplasms of adenoma and adenocarcinomas. A secondary objective was to examine the interaction of the bacterial mediated immune response with microRNA (miRNA) regulation. The elucidation of likely mechanisms whereby F. nucleatum may contribute to inflammatory mediated colorectal carcinogenesis will help to better understand the molecular pathways activated by this bacterium and where prevention and treatment strategies can be best targeted.

Research methods

Robust techniques were used for DNA quantification of F. nucleatum and RNA transcript measures of the inflammatory genes and miRNAs in normal and tumor tissues. For this purpose, we used TaqMan gene expression assays (Applied Biosystems, Foster City, CA, United States) with specific probes for each gene and miRNA for relative quantification. The reactions were analyzed using the StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, United States). Mutation testing of the KRAS gene was performed by direct sequencing and microsatellite instability (MSI) evaluation was performed using a multiplex PCR. In addition, we also used a bioinformatic tool ‘miRNA Data Integration Portal’ ([34] to build an miRNA:mRNA interaction network by using Cytoscape software (version 3.1.1)[36].

Research results

Ours results confirm the overabundance of F. nucleatum in adenoma and tumor neoplasms compared to their respective matched normal tissues, as previously found in several populations. We further suggest that this bacterial load increases the expression of TLR2 and TLR4 receptors and consequently of pro-inflammatory interleukins IL1B, IL6 and IL8. This immune-modulation of the inflammatory response to F. nucleatum colonic invasion also affects the expression of miRNA regulators of the inflammatory response. In particular, these miRNA:mRNA interactions network indicate a mechanism of colorectal carcinogenesis where altered expression of miR-34a, miR-135b, and miR-22, previously associated with CRC, occurs via a TLR2/TLR4 dependent response to F. nucleatum. In analyses stratified by tumor molecular characteristics, we observed that KRAS was more frequently mutated in tumors with F. nucleatum, and that an increased IL-8 expression was associated with MSI-high status. Therefore, more studies of gene function and regulation within the inflammatory pathways impacted by F. nucleatum invasion are needed, along with consideration of tumor molecular subtypes.

Research conclusions

Our findings reinforce the increasing invasion of F. nucleatum during the colorectal adenoma to cancer development. This appears to increase expression of pro-inflammatory mediators and dysregulation of miRNA expression, leading to a more carcinogenic microenvironment alongside genetic alterations such as KRAS mutation and MSI-high. Therefore, together with other studies, our results suggest that F. nucleatum is involved in CRC development through immune responses to inflammatory stresses. Further work is needed to functionally demonstrate these postulated tumorigenic mechanisms, and also for early CRC detection and diagnosis strategies using biomarkers of F. nucleatum presence or the consequent immune response.

Research perspectives

The intestinal microbiota is very diverse and important for the maintenance of epitelial homeostasis. Disturbances of this microbiome balance appears to be a major factor in CRC etiology. F. nucleatum has been implicated in recent years, by in vitro and in mouse models, as a carcinogenic bacterium through generation of a microenvironment conducive to cancer development. Considering that F. nucleatum has been found to be highly abundant in both adenoma and CRC neoplasms, it may have uses as a tissue or non-invasive biomarker in faeces (or possibly mouth-rinse samples) for CRC and the early detection of adenomas (which may help define a higher risk group for CRC development due to the presences of the bacterium). However, further investigations are needed to understand the molecular mechanisms in the immuno-inflammatory response to the increased invasion of this bacterium into developing neoplasms, and if this can promote genetic and epigenetic alterations that may culminate in CRC development.