Alterations in the host immune components, including Toll-like receptors (TLRs), through their prominent role in activating the innate and adaptive arms following infection, may influence the progress of the disease elicited by
TLR9 detects unmethylated CpG oligonucleotides present abundantly in bacterial DNA (Fukata and Abreu 2008). As TLR9 is located inside the intracellular endosomes, its activation requires intracellular transfer of unmethylated CpG oligonucleotides through endocytosis (Fűri et al. 2013). They are expressed by gastric epithelial cells and contribute considerably to immune recognition and signaling following
Different virulence agents of
The vacuolating cytotoxin (VacA) is another effective toxin produced by
The superoxide dismutase enzyme (SOD), encoded by the
The TLR9 plays a significant role in initiating the immune response following
We have excluded any participant who fulfilled one or more of the set exclusion criteria: the previous gastric surgery and the use of anti-
A peripheral blood sample of 10 ml was collected under complete aseptic precautions from each study participant for investigating TLR9 gene polymorphisms (TLR9 -1486T/C, rs187084 SNPs).
After three days, culture plates were examined for colonies where
The set of primers used to amplify the targeted gene was mentioned in Table I (Santos et al. 2012). The PCR was carried out using a master mix (Fermentas, Germany) that included 4 mM MgCl2, 0.4 mM of each dNTP, and 0.05 U/μl
Sequences of primer sets used.
Gene targeted | Sequence | Size of amplified product (bp) | Ref |
---|---|---|---|
F: 5′-AAGCTTTTAGGGGTGTTAGGGGTTT-3′ |
294 | Santos et al. 2012 | |
F: 5′-GATAACAGGCAAGCTTTTGAGG-3′ |
349 | Amin et al. 2019 | |
F: 5′-GCCCTGTGGCGTTTGATTTCC-3′ |
425 | Ryberg et al. 2008 | |
F: 5′-GCTCCAAGCATCACCAAAGACG-3′ |
603 | Ryberg et al. 2008 | |
F: 5′-CAATCGTGTGGGTTCTGGAGC-3′ |
678 | Ryberg et al. 2008 | |
F: 5′-ATGGAAATACAACAAACACAC-3′ |
259 | Harrison et al. 2017 | |
F: 5′-GGTCAAAATGCGGTCATGG-3′ |
290 | Harrison et al. 2017 | |
F: 5′-CATAACTAGCGCCTTGCAC-3′ |
352 | Harrison et al. 2017 | |
TLR9-1486T/C, rs187084 | 5′-TTCATTCATTCAGCCTTCACTCA-3′ |
490 | Roszak et al. 2012 |
For amplification of the
PCR reaction was run with thermal cycling conditions of 4 minutes at 95°C then 35 cycles each starting with 30 seconds at 95°C followed by 20 seconds at 60°C, and 30 seconds at 72°C to be ended with final extension for 5 minutes at 72°C to produce a DNA piece of 490 bp (Paradowska et al. 2016). The primers used were illustrated in Table I (Roszak et al. 2012).
Then the DNA products were digested using AflII restriction enzyme (Thermo Scientific, EU, Lithuania) by incubation for three hours at 37°C to yield one of three variants: two fragments of 192 bp, and 327 bp that indicated TT allele, or three fragments of 192 bp, 327 bp, and 490 bp that proved the presence of TC allele or an intact PCR fragment of 490 bp indicating the presence of CC allele (Paradowska et al. 2016).
This study included 106 subjects with gastric cancer and positive for
Demographic and histopathological data of the subjects included in the study.
Variable | Gastric cancer patients | Healthy study participants | |
---|---|---|---|
Age | |||
Years (mean ± SD) | 56.55 ± 8.63 | 53.27 ± 8.55 | 0.93 |
Sex | |||
Male | 63 (59.4) | 59 (55.7%) | 0.76 |
Female | 43 (40.6%) | 47 (44.3%) | |
Differentiation of the tumor | |||
Well differentiated tumor | 16 (15.1%) | NA | – |
Moderately differentiated tumor | 35 (33.0%) | ||
Poorly differentiated tumor | 55 (51.9%) |
Values are given as mean ± SD, or number (percentage)
NA – Not applicable
Of the 132 gastric cancer patients, 106 (80.3%) gave positive results for
Distribution of
Number | % | ||
---|---|---|---|
Positive | 106 | 80.3 | 0.000* |
Negative | 26 | 19.7 | |
Total | 132 | 100% |
– statistically significant
Distribution of virulence genes in
Virulence gene | N = 106 | % | |
---|---|---|---|
Positive | 96 | 90.6 | 0.000* |
Negative | 10 | 9.4 | |
Positive | 75 | 70.8 | 0.000* |
Negative | 31 | 29.2 | |
Positive | 88 | 83.0 | 0.000* |
Negative | 18 | 17.0 | |
Positive | 101 | 95.3 | 0.000* |
Negative | 5 | 4.7 | |
Positive | 101 | 95.3 | 0.000* |
Negative | 5 | 4.7 | |
Positive | 56 | 52.8 | 0.56 |
Negative | 50 | 47.2 | |
Positive | 45 | 42.5 | 0.12 |
Negative | 61 | 57.5 |
– statistically significant
We screened both the case and control groups for TLR9 -1486T/C, rs187084 polymorphism by PCR-RFLP. The obtained frequencies of genotypes of TLR9 -1486T/C, rs187084 in the healthy group were all on line with Hardy-Weinberg equilibrium. In gastric cancer patients, the frequencies of T and C alleles were 72 (34.0%) and 140 (66.0%), respectively, whereas in the control group, the T allele frequency was 122 (57.5%), and the C allele frequency was 90 (42.5%). We found that the frequency of the C allele in gastric cancer patients was significantly higher than in the control group (
Distribution of genotypes of TLR9 -1486T/C, rs187084 polymorphism in studied groups.
Genotype frequency | Healthy study participants N = 106 | Gastric cancer patients N = 106 | OR | 95% CI | ||||
---|---|---|---|---|---|---|---|---|
N | % | N | % | |||||
TT | 40 | 37.7 | 22 | 20.8 | 1.18 | 0.45–3.1 | 0.12 | 0.73 |
TC | 42 | 39.6 | 28 | 26.4 | 1.2 | 0.49–2.88 | 0.16 | 0.68 |
CC | 24 | 22.6 | 56 | 52.8 | 2.68 | 1.0–7.14 | 4.03 | 0.045* |
Genotype frequencies are presented in the form of absolute numbers with percentages
OR – Odds ratio;
CI – Confidence interval;
– statistically significant
The CC genotype of TLR9 -1486T/C, rs187084, when compared to TT + TC genotypes, demonstrated a significant relation with
The relation between virulence genes of
TLR9-1486T/C, rs187084 genotype | CC N = 56 | TT + TC N = 50 | |||
---|---|---|---|---|---|
Virulence gene | N | % | N | % | |
55 | 98.2 | 41 | 82.0 | 0.075 | |
53 | 94.6 | 22 | 44.0 | 0.021* | |
53 | 94.6 | 35 | 70.0 | 0.049* | |
54 | 96.4 | 47 | 94.0 | 0.88 | |
54 | 96.4 | 47 | 94.0 | 0.88 | |
35 | 62.5 | 21 | 42.0 | 0.048* | |
19 | 33.9 | 26 | 52.0 | 0.81 |
– statistically significant
We have reported a prevalence of
In our study, the prevalence of
TLR9 plays a vital role in recognizing
For TLR9 -1486T/C gene polymorphisms, we found that the frequency of CC genotype was significantly higher in gastric cancer patients than the control group (
In a gene assay conducted by Tao et al. (2007), the C allele of TLR9 -1486T/C down-regulates the expression of TLR9, which leads to deficient immune recognition and signaling in response to
Most studies have focused on either host or bacterial risk factors for developing gastric cancer; however, we explored the relationship between the studied virulence genes of
This study suggested that patients with CC genotype of TLR9 -1486T/C (rs187084) might be at higher risk for the development of gastric cancer. In addition, our results offered some evidence that the co-existence of CC genotype of TLR9 -1486T/C and