Breast cancer is the most common cancer among women in Ukraine [1]. Approximately from 8% to 20% are categorized as triple-negative breast cancer (TNBC) that does not overexpress a human epidermal growth factor receptor 2 (HER-2/neu), estrogen receptor (ER) and progesterone receptor (PR) [2]. Recent experiments have shown that toll-like receptors (TLRs) can enhance cancer cell progression, induce evasion of immune surveillance, and induce chemotherapeutic and metastasis [3]. TLRs polymorphisms may be involved in the process of breast carcinogenesis and play an important role in cancer development and treatment.
The aim of this study was to investigate TLR2(G753A), TLR4(C399T), TLR9(G2848A) polymorphisms and analyze their association with TNBC development.
In the present study, samples of peripheral blood of 63 patients with TNBC from age 24 to 76 years (52 ± 3.5 years) were used as a biological material to investigate polymorphisms of TLR2, 4 and 9 genes. After DNA extraction, we used polymerase chain reaction (PCR) method to make many copies of the TLR 2, 4 and 9 genes with specific primers. The PCR products of TLR2 were digested by restriction endonuclease SsiI (AciI) at 37 °C overnight and then analyzed by 3% agarose gel electrophoresis. Bands of 228, 75 and 40 bp corresponded to TLR2 G/G; 268, 228, 75, 40 bp bands were designated as heterozygous G/A individuals; a band of 268, 75 bp corresponded to the homozygous A/A genotype. The PCR products of TLR4 were digested by restriction endonuclease Hinf I. It was incubated overnight at 37°C and electrophoresed in 3% agarose gel to identify the TLR4 alleles on the basis of the respective allele size. Bands of 406 bp corresponded to TLR4 C/C; 406, 377, 29 bp bands were designated as heterozygous C/T individuals; a band of 377, 29 bp corresponded to the homozygous T/T genotype. The PCR products of TLR9 were digested by restriction endonuclease Bsh 1236 I (BstU I) at 37 °C overnight and then analyzed by 3% agarose gel electrophoresis. Bands of 135, 42 bp corresponded to TLR9 G/G; 177, 135, 42 bp bands were designated as heterozygous G/A individuals; a band of 177 bp corresponded to the homozygous A/A genotype.
According to the results, in the group of 56 patients with TNBC, were found 76.8% (43/56 patients) – with homozygous genotype of the wild type TLR2 allele (genotype G/G), 5.4% (3/56 patients) – with homozygous genotype of the mutant type allele (genotype A/A) and 17.9% (10/56 patients) with heterozygous genotype (genotype A/G). Patients with the A/A genotype of the TLR2 gene have significant increased risk (in 13.15 times) of TNBC development compared to the control ($\chi^{2}$ = 11.49; p = 0.003).
Our results indicated that TLR4(C399T) is not associated with risk of TNBC development, as all of the 60 patients had CC genotype - homozygous genotype of the wild type allele of the TLR4 gene.
According to the results, in the group of 59 patients with TNBC, were found 33.9% (9/59 patients) – with homozygous genotype of the wild type TLR9 allele (genotype G/G), 15.3% (20/59 patients) – with homozygous genotype of the mutant type allele (genotype A/A) and 30.5% (30/59 patients) with heterozygous genotype (genotype A/G). Patients with the A/A genotype of the TLR9 gene have significant increased risk (in 9.77 times) of TNBC development compared to the control ($\chi^{2}$ = 95.94; p $\leq$ 0.0001).
We established that presence of TLR2(G753A) and TLR9(G2848A) polymorphisms have an increased risk of TNBC development and may be recommended to the diagnostic marker in the primary screening of BC. Our results showed that TLR4(C399T) is not associated with the TNBC development.