Involvement of toll-like receptor 9 polymorphism in cervical cancer development

The role played by the polymorphism located in Toll-like Receptor 9 (TLR9) as a risk factor of cervical cancer remains elusive. Therefore, we studied the association of the TLR9 −1486 T/C (rs187084) and C2848T (rs352140) polymorphisms with cervical cancer. The TLR9 −1486 T/C and C2848T polymorphism was genotyped in 426 patients and 460 unrelated healthy females from the Polish population. Logistic regression analysis adjusting for age, pregnancy, oral contraceptive use, tobacco smoking, and menopausal status showed that both the TLR9 −1486 T/C and C2848T polymorphisms could be a genetic risk factor for cervical cancer. For the TLR9 −1486 T/C polymorphism, the adjusted OR for patients with the C/T genotype versus T/T genotype was 1.371 (95 % CI 1.021–1.842, p = 0.0361), the adjusted OR for the C/C genotype vs the T/T genotype was 1.300 (95 % CI 1.016–1.507, p = 0.0096), and the adjusted OR for the C/T or C/C genotype vs the T/T genotype was 1.448 (95 % CI 1.099–1.908, p = 0.0083). For the C2848T polymorphism, the adjusted OR for patients with the C/T genotype vs C/C genotype was 1.443 (95 % CI 1.019–2.043, p = 0.0380), the adjusted OR for the T/T genotype vs the C/C genotype was 1.237 (95 % CI 1.016–1.507, p = 0.0328), and the adjusted OR for the T/C or T/T genotype vs the C/C genotype was 1.345 (95 % CI 0.976–1.855, p = 0.0700). Our studies suggest that the TLR9 −1486 T/C and C2848T polymorphisms may be a genetic risk factor for cervical cancer.

patterns and they play an elementary role in the innate immune response [14].
There are two studies on the association of the TLR9 1486 T/C (rs187084) and C2848T (rs352140) polymorphisms with cervical cancer in various populations, however, the results are inconsistent [25,26]. Therefore, we aimed to study whether TLR9 -1486 T/C (rs187084) and C2848T (rs352140) can be a genetic risk factor of cervical cancer in the Polish population.

Patients and controls
The patient group was composed of 426 women with histologically determined cervical carcinoma according to the International Federation of Gynecology and Obstetrics (FIGO). All women were enrolled between April 2007 and July 2011 at the Department of Radiotherapy, Greater Poland Cancer Center in Poznan, Poland (Table 1). The controls included four hundred sixty unrelated healthy female volunteers who were matched by age to the patients (Table 1). Information about pregnancy, oral contraceptive use, tobacco smoking, and menopausal status was obtained as part of the patient history. Patients and controls were Caucasian, enrolled from the Wielkopolska area of Poland. All subjects provided written informed consent. The study was approved by the Local Ethical Committee of Poznan University of Medical Sciences.
The PCR-amplified fragments 565 bp in length bearing the TLR9 -1486 T/C (rs187084) polymorphism were digested with restriction enzyme Afl II (C/TTAAG) New England BioLabs, (Ipswich, USA). The TLR9 T allele was cleaved into 416 and 149 bp fragments, whereas the TLR9 C allele remained uncut. The PCR-amplified fragments 360 bp in length corresponding to the TLR9 C2848T a Age at first diagnosis polymorphism were subjected to digestion with the endonuclease BstUI (CG/CG) New England BioLabs, (Ipswich, USA). The TLR9 C allele was cleaved into 227 and 133 bp fragments, whereas the TLR9 T allele remained uncut. DNA fragments were separated by electrophoresis on 3 % agarose gel and visualized by ethidium bromide staining. The TLR9 1486 T/C and C2848T polymorphism was confirmed by repeated PCR-RFLP. Moreover, the restriction analysis was confirmed by commercial sequencing analysis.

Statistical analysis
The distribution of genotypes in patients and controls was examined for deviation from Hardy-Weinberg equilibrium using exact and log likelihood ratio v 2 tests [http://ihg. gsf.de/cgi-bin/hw/hwa1.pl]. The polymorphism was tested for association with cervical cancer using the v 2 test for trend (p trend ). The v 2 test was employed to examine differences in genotypic and allelic distribution between patients and controls. The odds ratio (OR) and 95 % confidence intervals (95 % CI) were calculated. Unconditional logistic regression analysis was used to adjust for the effect of confounders such as age, pregnancy, oral contraceptive use, tobacco smoking, and menopausal status. A p value of \0.05 was considered statistically significant.

Results
Prevalence of the TLR9 -1486 T/C polymorphism in women with cervical cancer There was a higher frequency of the TLR9 CC genotype in women with cervical cancer compared to healthy individuals, which was 0.19 and 0.14, respectively ( Table 2). We also found increased TLR9 C/T heterozygote frequency in patients than in controls, which was 0.48 and 0.44, respectively (Table 2). There was also an increased TLR9 C allele frequency in patients than in controls, which was 0.43 and 0.36, respectively ( Table 2). The p value of the v 2 test of the trend observed for the TLR9 -1486 T/C polymorphism was statistically significant (p trend = 0.0042). Logistic regression analysis showed a significant contribution of the TLR9 -1486 T/C polymorphism to cervical cancer ( Table 2). The adjusted OR for patients with the C/T genotype vs T/T genotype was 1.371 (95 % CI 1.021-1.842, p = 0.0361), the adjusted OR for the C/C genotype vs the T/T genotype was 1.300 (95 % CI 1.016-1.507, p = 0.0096), and the adjusted OR for the C/T or C/C genotype vs the T/T genotype was 1.448 (95 % CI 1.099-1.908, p = 0.0083) ( Table 2).
Prevalence of the TLR9 C2848T polymorphism in women with cervical cancer We observed an increased frequency of the TLR9 TT genotype in patients than controls, which was 0.26 and 0.22, respectively ( Table 2). The TLR9 C/T heterozygote frequency in women with cervical cancer was also increased compared to healthy individuals and amounted to 0.54 and 0.51, respectively ( Table 2). We also observed a higher TLR9 T allele frequency in patients then healthy individuals, which was 0.53 and 0.48, respectively ( Table 2). The p value of the v 2 test of the trend observed for the TLR9 C2848T polymorphism was statistically significant (p trend = 0.0449). Logistic regression analysis showed a significant association of the TLR9 C2848T polymorphism with cervical cancer ( Table 2). The adjusted OR for patients with the C/T genotype vs C/C genotype was 1.443 (95 % CI 1.019-2.043, p = 0.0380), the adjusted OR for the T/T genotype vs the C/C genotype was 1.237 (95 % CI 1.016-1.507, p = 0.0328), and the adjusted OR for the T/C or T/T genotype vs the C/C genotype was 1.345 (95 % CI 0.976-1.855, p = 0.0700) ( Table 2).

Discussion
TLR9 plays a crucial role in pathogen recognition and activation of innate immunity [15,18]. Stimulation of TLR9 activates human B cells and plasmacytoid dendritic cells, causing T helper-1 type immune responses and antitumor responses [18,[27][28][29][30][31]. The role of the TLR9 pathway in anticancer treatment has been considered in animal models and patients with renal carcinoma, malignant melanoma, and non-Hodgkin's lymphoma [27][28][29][30][31]. Moreover, the significance of the TLR9 pathway has been demonstrated in vaccine treatment of cervical carcinoma in murine model [28]. Mansilla et al. [28] demonstrated that intratumoral administration of EDA-HPVE7 fusion protein in combination with TLR9 ligand CpG-B was able to eradicate large established cervical tumors. HPV infects primitive basal keratinocytes; however, its abundant expression and viral assembly take place only in the upper layers of the stratum spinosum and granulosum of squamous epithelia [32]. Keratinocytes bear TLR9, and stimulation of TLR9 may result in the production of a spectrum of mediators influencing the function of immune cells [21]. Some studies have suggested that changes occur in TLR9 expression during cervical carcinogenesis [11,13]. Recently, Hasimu et al. (2007) found that the expression of TLR9 can be upregulated by HPV16 infection in CIN and in cervical squamous carcinoma cells [11]. They also suggested that TLR9 may play important roles in the development and progression of CIN and cervical carcinoma [11]. Adding to the above findings are those of Hasan et al. [13], who demonstrated that HPV16 infection of human primary keratinocytes reduced TLR9 transcription and lead to a functional loss of TLR9regulated pathways. This may suggest that polymorphisms of TLR9 that modulate the expression of TLR9 may have an effect on the etiopathogenesis of cervical cancer [33].
We observed a contribution of the TLR9 -1486 T/C (rs187084) and C2848T (rs352140) polymorphisms to the risk of cervical cancer in a Polish population. Recent studies conducted by Chen et al. [25] demonstrated that the TLR9 -1486 T/C (rs187084) polymorphism, located in the LD block with rs352140, was associated with a significantly increased risk of cervical cancer. In contrast, Pandey et al. [26] showed that the TT genotype of TLR9 (rs352140) displayed borderline significance in increased risk for advanced cervical cancer in a North India population.
These differences in the effect of TRL9 polymorphism on the susceptibility to cervical cancer development between our and Hindu populations may result from racial heterogeneity, the size of the studied groups, and the action of distinct behavioral and environmental factors [33].
The role of the rs352140 polymorphism on TLR9 expression has been suggested by Kikuchi et al. [24],who demonstrated that the TLR9 2848 TT genotype was associated with a higher expression of TLR9 and an increased frequency of IgM ? B cells. It has been believed that chronic inflammation may lead to cancer development and progression (33). The increased expression of the TLR9 2848 T variant in precursor malignant lesion cells combined with infection by various pathogens might support inflammation and cervical cancer development (33).
Our studies suggest that the C2848T (rs352140) polymorphism might be a risk factor of cervical cancer in Polish women. Our genetic evaluation is the first in a Caucasian cohort; therefore this study should be replicated in a larger and independent cohort.