Case Control Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Nov 15, 2019; 11(11): 998-1010
Published online Nov 15, 2019. doi: 10.4251/wjgo.v11.i11.998
Toll-like receptor 9 polymorphisms and Helicobacter pylori influence gene expression and risk of gastric carcinogenesis in the Brazilian population.
Manoela Dias Susi, de Matos Lourenço Caroline, Lucas Trevizani Rasmussen, Spencer Luis Marques Payão, Ana Flávia Teixeira Rossi, Ana Elizabete Silva, Juliana Garcia de Oliveira-Cucolo
Manoela Dias Susi, de Matos Lourenço Caroline, Department of Graduate-Level Research, USC-Sacred Heart University, Bauru 17011-970, SP, Brazil
Lucas Trevizani Rasmussen, Spencer Luis Marques Payão, Department of Genetics and Molecular Biology, FAMEMA-Marilia Medical School, Marília 17519-030, SP, Brazil
Ana Flávia Teixeira Rossi, Ana Elizabete Silva, Department of Biology, São Paulo State University-UNESP, São José do Rio Preto 15054-000, SP, Brazil
Juliana Garcia de Oliveira-Cucolo, Department of Molecular, Biological and Genetics and Molecular Biology Research Unit – UPGEM, Faculty of Medicine of São José do Rio Preto – FAMERP, São José do Rio Preto 15090-000, SP, Brazil.
Author contributions: Oliveira-Cucolo JG and Silva AE conceived and designed the experiments; Susi MD, de Matos Lourenço C, Rossi AFT and Oliveira-Cucolo JG performed the experiments; Oliveira-Cucolo JG, Silva AE and Rossi AFT analyzed and interpreted the data; Rasmussen LT, Payão SLM and Silva AE contributed with the collection of samples/reagents/materials and analysis tools; Oliveira-Cucolo JG, Rossi AFT and Silva AE drafted the manuscript and revised it; All of the authors have approved the final version of manuscript for publication.
Supported by The São Paulo Research Foundation (FAPESP), NO. 2013/14022-6 and NO. 2014/17716-1.
Institutional review board statement: This study was approved by the research ethics committee of USC-Sacred Heart University.
Informed consent statement: Informed consent was obtained from each patient.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: Participants gave written informed consent for data sharing.
STROBE statement: The manuscript was prepared and revised according to the STROBE Statement-checklist of items.
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: Ana Elizabete Silva, PhD, Adjunct Professor, Department of Biology, São Paulo State University-UNESP, Rua Cristóvão Colombo 2265, São José do Rio Preto 15054-000, SP, Brazil. ae.silva@unesp.br
Telephone: +55-17-32212384 Fax: +55-17-322212390
Received: March 14, 2019
Peer-review started: March 14, 2019
First decision: May 9, 2019
Revised: July 26, 2019
Accepted: August 27, 2019
Article in press: August 27, 2019
Published online: November 15, 2019
Abstract
BACKGROUND

Toll-like receptors (TLRs) are the first line of host defense, and are involved in Helicobacter pylori (H. pylori) recognition and activation of both inflammatory and carcinogenic processes. The presence of single nucleotide polymorphisms (SNPs) in genes that activate the immune response may modulate the risk of precancerous lesions and gastric cancer (GC). Among them, Toll-like receptor 9 (TLR9) polymorphisms have emerged with a risk factor of infectious diseases and cancer, however the studies are still inconclusive.

AIM

To evaluate whether TLR9 rs5743836 and rs187084 SNPs contribute to the risk of gastric carcinogenesis, and its influence on mRNA expression.

METHODS

A case-control study was conducted to evaluate two TLR9 SNPs (TLR9-1237 TC-rs5743836 and TLR9-1486 CT-rs187084) in chronic gastritis (CG) and GC patients. A total of 609 DNA samples of peripheral blood [248 CG, 161 GC, and 200 samples from healthy individuals (C)] were genotyped by polymerase chain reaction-restriction fragment length polymorphism. All samples were tested for the H. pylori infection using Hpx1 and Hpx2 primers. Quantitative polymerase chain reaction by TaqMan® assay was used to quantify TLR9 mRNA from fresh gastric tissues (48 GC, 26 CG, and 14 C).

RESULTS

For TLR9-1237, the TC + CC or CC genotypes were associated with a higher risk of GC than C [recessive model odds ratio (OR) = 5.01, 95% confidence interval (CI): 2.52-9.94, P < 0.0001], and the CG (recessive model OR =4.63; 95%CI: 2.44-8.79, P < 0.0001) groups. For TLR9-1486, an association between the CT + TT genotypes and increased risk of both GC (dominant model OR = 2.72, 95%CI: 1.57-4.72, P < 0.0001) and CG (dominant model OR = 1.79, 95%CI: 1.15-2.79, P = 0.0094) was observed when compared to the C group. Moreover, the presence of TLR9-1237 TC/CC + TLR9-1486 CC genotypes potentiate the risk for this neoplasm (OR = 18.57; 95%CI: 5.06-68.15, P < 0.0001). The TLR9 mRNA level was significantly higher in the GC group (RQ = 9.24, P < 0.0001) in relation to the CG group (RQ = 1.55, P = 0.0010) and normal mucosa (RQ = 1.0). When the samples were grouped according to the polymorphic genotypes and the presence of H. pylori infection, an influence of TLR9-1237 TC + CC polymorphic genotypes (P = 0.0083) and H. pylori infection (P < 0.0001) was observed on the upregulation of mRNA expression.

CONCLUSION

Our findings show that TLR9 rs5743836 and rs187084 polymorphisms are associated with a higher risk of carcinogenesis gastric, and that TLR9 mRNA levels can be modulated by TLR9-1237 TC + CC variant genotypes and H. pylori infection.

Keywords: Toll-like receptor 9, Helicobacter pylori, Gastric cancer, Chronic gastritis, Polymorphisms, Gene expression

Core tip: This study investigated the influence of Toll-like receptor 9 (TLR9) polymorphisms on mRNA and Helicobacter pylori (H. pylori) infection in gastric cancer samples, and the association of these single nucleotide polymorphisms with the risk of developing this neoplasm. Increased expression of TLR9 in tumor tissue was observed compared with chronic gastritis and normal tissue. Moreover, when samples were grouped according to H. pylori presence, an upregulation of TLR9 mRNA expression was observed. Thus, both H. pylori infection as well as functional TLR9 polymorphisms may change gene expression levels, accentuating inflammation and aggravating the development of gastric cancer.