Case Control Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. May 15, 2020; 12(5): 535-548
Published online May 15, 2020. doi: 10.4251/wjgo.v12.i5.535
Characterization and strong risk association of TLR2 del -196 to -174 polymorphism and Helicobacter pylori and their influence on mRNA expression in gastric cancer
Caroline de Matos Lourenço, Manoela Dias Susi, Mariah Cristina Antunes do Nascimento, Vilson Serafim Junior, Ana Paula Simedan Vila, Gabriela Helena Rodrigues-Flemming, Eny Maria Goloni-Bertollo, Ana Elizabete Silva, Juliana Garcia de Oliveira-Cucolo
Caroline de Matos Lourenço, Manoela Dias Susi, Department of Graduate-Level Research, Sacred Heart University, Bauru, São Paulo 17011-970, Brazil
Mariah Cristina Antunes do Nascimento, Vilson Serafim Junior, Ana Paula Simedan Vila, Gabriela Helena Rodrigues-Flemming, Eny Maria Goloni-Bertollo, Juliana Garcia de Oliveira-Cucolo, Department of Molecular Biology, São José do Rio Preto School of Medicine, São José do Rio Preto, São Paulo 15090-000, Brazil
Ana Elizabete Silva, Department of Biology, São Paulo State University, São José do Rio Preto, São Paulo 15054-000, Brazil
Author contributions: Oliveira-Cucolo JG and Silva AE conceived and designed the experiments; Lourenço CM, Susi MD, Serafim Junior V and Vila APS performed the experiments; Nascimento MCA provided technical support; Oliveira-Cucolo JG, Serafim Junior V and Rodrigues-Flemming GH analyzed and interpreted the data; Silva AE and Goloni-Bertollo EM contributed to the collection of samples/reagents/materials and analysis tools; Oliveira-Cucolo JG, Rodrigues-Flemming, Goloni-Bertollo EM and Silva AE drafted and revised the manuscript; All of the authors approved the final version of the manuscript for publication.
Supported by the São Paulo Research Foundation, No. 2013/14022-6 and No. 2014/17716-1.
Institutional review board statement: This study was approved by the local Research Ethics Committee (registration number: 382.514).
Conflict-of-interest statement: The authors declare no conflicts of interest.
Informed consent statement: The participants provided written informed consent for data sharing.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (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: Ana Elizabete Silva, PhD, Adjunct Professor, Department of Biology, São Paulo State University, Rua Cristóvão Colombo, 2265, São José do Rio Preto, São Paulo 15054-000, Brazil.
Received: December 13, 2019
Peer-review started: December 13, 2019
First decision: January 14, 2020
Revised: January 30, 2020
Accepted: April 4, 2020
Article in press: April 4, 2020
Published online: May 15, 2020
Research background

Helicobacter pylori (H. pylori) infection is a carcinogen for gastric cancer (GC), and Toll-like receptors are involved in recognition and activation of the inflammatory response for this bacterium. The presence of single nucleotide polymorphism (SNP) genes responsible for activating the innate immunity may influence the risk of precancerous lesions and GC, among them of which include TLR2 polymorphisms. GC is the fifth most common cancer worldwide, mortality rates are still high. In Brazil, GC is the fourth most frequent type of cancer in men, and the sixth in women, with an estimated incidence of 21230 new cases in 2020.

Research motivation

Considering the inconsistent results in the literature, as well as the importance of these receptors in immune response and for the susceptibility to inflammatory diseases and cancer, new studies are needed. The Brazilian population is highly mixed; thus it becomes important to confirm the real role among the factors that influence changes in the recognition of H. pylori and gastric carcinogenesis.

Research objectives

The aim of this study was to evaluate whether the TLR2 19216T/C (rs3804099) and TLR2 -196 to -174 ins/del (rs111200466) polymorphisms contribute to gastric carcinogenesis in the Brazilian population. In addition, we also evaluate the influence of both polymorphisms and H. pylori infection on TLR2 mRNA expression. The results may highlight important polymorphisms that act on gastric carcinogenesis.

Research methods

A case-control study was conducted to evaluate two TLR2 SNPs (TLR2 19216T/C -rs3804099 and TLR2 -196 to -174 ins/del - rs111200466) in CG and GC patients. A total of 854 DNA samples of peripheral blood [269 CG, 202 GC, and 383 samples from healthy individuals (C)] were genotyped by allele-specific PCR or restriction fragment length polymorphism (RFLP)-PCR. Quantitative polymerase chain reaction by TaqMan® assay was used to quantify TLR2 mRNA from fresh gastric tissues (48 GC, 26 CG, and 14 C).

Research results

The data showed that for the TLR2 -196 to -174 polymorphism, the ins/del and del/del genotypes were associated with a higher risk of GC compared with the C and CG groups. In contrast, TLR2 19216T/C was associated with a protective effect in the GC group compared to the C group. Regarding the association of polymorphisms with H. pylori infection, for the TLR2 -196 to -174 ins/del polymorphism, an association was observed with H. pylori-positive, while TLR2 19216T/C was associated with protection against H. pylori infection. TLR2 mRNA levels were significantly higher in the GC group compared to the CG group and normal mucosa. In addition, when the samples were grouped according to polymorphic genotypes and the presence of H. pylori, the two SNPs (TLR2 -196 to -174del and TLR2 19216 C alleles) and H. pylori infection influenced TLR2 mRNA expression.

Research conclusions

Our findings highlight that the polymorphisms of the TLR2 -196 to -174 ins/del and TLR2 19216 T/C receptors are associated with gastric cancer (an increased risk and a protective effect, respectively) and H. pylori infection, and therefore may act as a potential factor in the progression of gastric carcinogenesis. TLR2 mRNA expression levels are upregulated in gastric cancer tissues and are influenced by the TLR2 -196 to -174 del variant allele, the TLR2 19216 T wild-type allele and H. pylori infection. Considering that most cases of GC have a good prognosis and are treatable when diagnosed at an early stage, it is of the utmost importance to establish molecular markers capable of identifying risk groups and providing early diagnosis in individuals with increased risk of developing this neoplasm. Thus, polymorphisms in genes that affect its expression, such as TLR2, could have an effect on the development and clinical manifestation of disease.

Research perspectives

The pattern of the host’s immune response associated with genetic and environmental factors are essential for understanding the pathology of gastric cancer. Overall, our results indicate that the TLR2 gene plays an important role in gastric carcinogenesis, highlighting the importance of the TLR2 -196 to -174 del and TLR2 19216 T polymorphisms in increasing gene expression and H. pylori infection, possibly triggering a stronger inflammatory response, which in turn enhances the risk of tumor progression. In the future, it would be important to increase the biopsies collected during the endoscopic evaluation to quantify TLR2 mRNA levels, and investigate another polymorphism in the TLR2 gene (rs3804100, rs7696323, and rs10116253), described in the literature as associated with cancer, but not yet analyzed in our Brazilian population.