Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 7, 2015; 21(25): 7730-7741
Published online Jul 7, 2015. doi: 10.3748/wjg.v21.i25.7730
TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer
Marcela Alcântara Proença, Juliana Garcia de Oliveira, Aline Cristina Targa Cadamuro, Maysa Succi, João Gomes Netinho, Eny Maria Goloni-Bertolo, Érika Cristina Pavarino, Ana Elizabete Silva
Marcela Alcântara Proença, Aline Cristina Targa Cadamuro, Maysa Succi, Ana Elizabete Silva, Department of Biology, UNESP, São Paulo State University, São José do Rio Preto 15054-000, SP, Brazil
Juliana Garcia de Oliveira, USC- Sacred Heart University, Pró-Reitoria de Pesquisa e Pós Graduação, Bauru 17011-160, SP, Brazil
João Gomes Netinho, Department of Surgery, School of Medicine, FAMERP, São José do Rio Preto 15090-000, SP, Brazil
Eny Maria Goloni-Bertolo, Érika Cristina Pavarino, UPGEM, School of Medicine, FAMERP, São José do Rio Preto 15090-000, SP, Brazil
Author contributions: Proença MA planned and conducted the study, collected and interpreted the data, drafted and wrote the manuscript; de Oliveira JG collected data on genotyping of TLR2 and TLR4 polymorphisms in the control group; Cadamuro ACT collected data on immunohistochemistry of both TLR2 and TLR4 proteins; Succi M collected data on CRC samples; Netinho JG collected data on CRC samples; Goloni-Bertolo EM and Pavarino ÉC planned the study; Silva AE conceived and planned the study and critically revised the manuscript.
Supported by Grants from Brazilian agencies FAPESP, No. 2012/15036-8; and CNPq, No. 304870/2012-9.
Ethics approval: This study was reviewed and approved by the Ethics in Research Committee IBILCE/UNESP, nº027/11 (protocol: 0009.0.229.000-11).
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: All 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/
Correspondence to: Ana Elizabete Silva, PhD, Department of Biology, UNESP, São Paulo State University, Rua Cristóvão Colombo, 2265, São José do Rio Preto 15054-000, SP, Brazil. anabete@ibilce.unesp.br
Telephone: +55-17-32212384 Fax: +55 17-32212390
Received: October 16, 2014
Peer-review started: October 18, 2014
First decision: December 2, 2014
Revised: January 3, 2015
Accepted: February 12, 2015
Article in press: February 13, 2015
Published online: July 7, 2015
Abstract

AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk.

METHODS: The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies.

RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0.26).

CONCLUSION: Our findings suggest that TLR2-196 to -174del polymorphism increases TLR2 mRNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility.

Keywords: Toll-like receptor 2, Toll-like receptor 4, Colorectal cancer, Protein expression, Gene expression, Genetic polymorphisms

Core tip: This study investigated the influence of the toll-like receptor (TLR)2 and TLR4 functional polymorphisms on mRNA and protein expression levels in colorectal cancer samples and the association of these polymorphisms with the risk of developing this neoplasm. Increased expression of TLR2 (mRNA and protein) in tumor tissue was observed compared with adjacent normal tissue. Moreover, for the first time, the polymorphism TLR2-196 to -174del was associated with a higher risk of developing this type of cancer, and TLR2-196 to -174del allele carriers showed mRNA relative expression approximately two times higher than wild-genotype carriers. Thus, functional polymorphism in TLR2 may change gene expression levels, accentuating inflammation and aggravating the development of colorectal cancer.