Characterization and strong risk association of TLR2 del -196 to -174 polymorphism and Helicobacter pylori and their influence on mRNA expression in gastric cancer

Abstract

Background: Toll-like receptor-2 (TLR2) is responsible for recognizing Helicobacter pylori (H. pylori) and activating the immune response. Polymorphisms in TLR2 may modulate gastric carcinogenesis.

Aim: 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, and to determine the influence of both polymorphisms and H. pylori infection on TLR2 mRNA expression.

Methods: DNA was extracted from 854 peripheral blood leukocyte or gastric tissue samples [202 gastric cancer (GC), 269 chronic gastritis (CG), and 383 control/healthy (C)] and 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 levels in fresh gastric tissues (48 GC, 36 CG, and 14 C).

Results: Regarding the TLR2 -196 to -174 polymorphism, the ins/del and del/del genotypes were associated with a higher risk of GC by comparison with the C in all of the analyzed inheritance models (codominant, dominant, recessive, overdominant and log-additive; P < 0.0001). Similarly, an increased risk was observed when comparing the GC and CG groups [codominant (P < 0.0001), dominant (P < 0.0001), recessive (P = 0.0260), overdominant (P < 0.0001) and log-additive (P < 0.0001)]. In contrast, TLR2 19216T/C was associated with a protective effect in the GC group compared to the C group [dominant (P = 0.0420) and log-additive (P = 0.0300)]. Regarding the association of polymorphisms with H. pylori infection, individuals infected with H. pylori and harboring the TLR2 -196 to -174 ins/del polymorphism had an increased risk of gastric carcinogenesis [codominant (P = 0.0120), dominant (P = 0.0051), overdominant (P = 0.0240) and log-additive (P = 0.0030)], while TLR2 19216T/C was associated with a protective effect [codominant (P = 0.0039), dominant (P < 0.0001), overdominant (P = 0.0097) and log-additive (P = 0.0021)]. TLR2 mRNA levels were significantly increased in the GC group (median RQ = 6.95) compared to the CG group (RQ = 0.84, P < 0.0001) and to the normal mucosa group (RQ = 1.0). In addition, both H. pylori infection (P < 0.0001) and the presence of the polymorphic TLR2 -196 to -174del (P = 0.0010) and TLR2 19216 C (P = 0.0004) alleles influenced TLR2 mRNA expression.

Conclusion: The TLR2 -196 to -174 ins/del and TLR2 19216 T/C polymorphisms are strongly associated with GC. TLR2 mRNA expression levels are upregulated in neoplastic tissues and influenced by both the presence of H. pylori and variant genotypes.

Keywords: Chronic gastritis; Gastric cancer; Gene expression; Helicobacter pylori; Polymorphisms; Toll-like receptor 2.

Figure 1

Relative gene expression levels of TLR2. A: Comparison between gastric cancer (GC) and chronic gastritis groups (CG), ^aP < 0.0001; and GC or CG groups with samples of normal mucosa (RQ = 1 represented for dashed line), ^bP < 0.0001 and P = 0.9387, respectively. B: Individual representation of RQ for each sample of GC group; C: Individual representation of relative quantification (RQ) for each sample of CG group. Significant difference, ^{a, b}P < 0.05. RQ represented by median with interquartile range. Mann-Whitney and Wilcoxon Signed statistical test.

Figure 2

Relative gene expression levels of TLR2 according to Helicobacter pylori infection. A: Comparison between Helicobacter pylori (H. pylori)-positive and H. pylori-negative groups, ^aP < 0.0001; H. pylori-positive or H. pylori-negative groups with samples of normal mucosa (relative quantification (RQ) = 1 represented for dashed line), ^bP < 0.0001 and P = 0.4305, respectively. B: Individual representation of RQ for each sample in H. pylori-positive group; C: Individual representation of RQ for each sample in H. pylori-negative group. ^{a, b}P < 0.05. RQ represented by median with interquartile range. Mann-Whitney and Wilcoxon Signed statistical test.

Figure 3

Relative gene expression levels of TLR2 according to polymorphic genotypes. A: Comparison between TLR2-196 to -174ins/ins (wild-type) and ins/del + del/del (polymorphic) in gastric cancer (GC), P = 0.0010 and chronic gastritis (CG), P = 0.5334 groups. B: TLR2-19216 TT (wild type) and TC + CC (polymorphic) in GC, P = 0.0004 and CG, P = 0.8827 groups. Significant difference, ^aP < 0.05. Relative quantification (RQ) represented by median with interquartile range. Mann-Whitney U statistical test.