Introduction

Cervical cancer remains the second-most common cause of cancer-related deaths in women worldwide, with about 450,000 new cases diagnosed every year [5, 18]. Although it is the most common female gynaecological malignancy in India, accounting for 26% of female cancers, with 90,000 women developing the disease every year [27]. Human papillomavirus (HPV) is one of the most common causes of sexually transmitted disease in both men and women worldwide [8]. More than 200 types of HPV have been recognized on the basis of DNA sequence data showing genomic differences. Eighty-five HPV genotypes are well characterized [36]. Based on their association, HPVs can also be grouped to high-risk and low-risk HPV types. Low-risk HPV types include types 6, 11, 42, 43, and 44. High-risk HPV types include 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70. Incorporated in the high-risk group are some HPV types that are less frequently found in cancers but are often found in squamous intraepithelial lesions (SILs) [10]. Also, human papillomaviruses are well known oncogenic dsDNA viruses linked to cervical cancer, and associated with the high-risk group of viral oncogenes E6 and E7 [35]. Among the high-risk HPVs, HPV 16 and HPV 18 are associated with 70% of all cervical carcinomas, and HPV type 16 correlates to 50% of cervical carcinomas [6, 13]. Among anti-inflammatory strategies, both at systemic and local level, the use of the IL-1 receptor antagonist (IL-1RA) has received vast attention. IL-1RA belongs to the IL-1 family, which consists of three linked genes mapping within a 430 kb region of the long arm of chromosome 2 in humans, encoding the secreted lycoproteins IL-1b, IL-1a, and IL-1Ra. All three molecules bind to IL-1 receptors [11]. IL-1Ra inhibits IL-1 by acting as a competitive receptor antagonist with no detectable agonistic activity, thus representing a natural powerful mechanism to control IL-1 dependent responses and avoid pathological derangement [21, 9]. The polymorphic gene that encodes IL-1RA seems to play an important role in the development of various diseases [33]. The IL-1RA gene is also polymorphic due to a variable number (2–6) of tandem repeat of 86 bp (VNTR) within its second intron [30]. Our basic objective of this study was to provide the relationship between polymorphism of lL-lRA (VNTR) gene and susceptibility to cervical cancer in North Indian population and tried to reveal the correlation between this genotype and HPV types 16 and 18 distribution.

Materials and methods

Patient and control

The case−control study involved collection of peripheral blood samples (2–5 ml) of 350 North Indian subjects. The 150 cases were newly diagnosed, previously untreated, and histologically confirmed cervix cancer patients. The samples were collected from Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, and Government Medical College (GMC), Chandigarh. The control peripheral blood samples (n = 200) were collected from these institutes with no history of cancer or pre-cancer.

Informed consent was obtained from all the cases and controls. Detailed data regarding age, use of oral contraceptive, education, menarche, menopausal status, number of children, age at marriage and birth of first child, cigarette smoking history, and spouse’s smoking history were also obtained.

DNA extraction

Genomic DNA was extracted from EDTA anti-coagulated peripheral blood according to a standard proteinase K digestion and phenol chloroform extraction method [24].

IL-1RA polymorphism analysis

The oligonucleotide primers flanking the 86 bp repeat region in intron 2 of IL-1RA were used (Table 1). Amplification reactions were carried out in a volume of 25 μl containing 100 ng genomic DNA, 0.25 mmol/l dNTPs (MBI Fermentas, Burlington, Ontario, Canada), 2 mmol/l MgCl2, 10 mM Tris–HCL (pH  8.3), 50 mM KCl, 1.5 U of Taq polymerase (MBI Fermentas), and 0.3 mmol/l of each primer (Sigma-Aldrich, Bangalore, India). PCR conditions included an initial denaturing step at 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 70°C for 2 min; and a final extension at 70°C for 4 min. Using this PCR strategy, the common allele (allele 1) generated a 410 bp band (including four copies of an 86 bp repeat). The uncommon alleles generated a 240 bp band (two copies of the same repeat; allele 2) a 500 bp band (five copies of the same repeat; allele 3), and a 325 bp band (allele 4). PCR products were resolved on a 3% agarose gel and stained with ethidium bromide (Sigma-Aldrich, USA)

Table 1 Primer sequence and length of PCR product
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HPV detection and typing

For HPV detection and typing to identify the HPV genome, general primers GP5 and GP6 with β-globin were used (Table 1), as described by Papadakis et al. [23]. β-globin was used as an internal standard. A total of 100 ng DNA from each patient was amplified by PCR containing 10 pmol of each primer, 2.5 mM of MgCl2, 0.2 mM each dNTPs, 1 U Taq polymerase in final (50 μl). The mixture was heated at 94°C for 3 min and samples were amplified for 40 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s; followed by elongation at 72°C for 5 min. PCR products were expected to be 139–145 bp, depending on HPV type [28]. HPV typing of all HPV-positive samples was carried out using multiplex PCR. Specific pairs of primers were used to simultaneously amplify regions of HPV types 16 and 18 in the same reaction tube, giving different lengths of amplified DNA. To establish type specificity of primer-directed amplification, PCR products were analyzed by electrophoresis in a 2% agarose gel, stained with ethidium bromide and observed under UV light transilluminator.

Statistical analysis

Age, age at marriage and at birth of first child, age at menarche and menopause, HPV status (positive, negative), and genotype of lL-lRA gene were tabulated for cases and controls (Table 2). Cases were further categorized into histological and clinical subtypes for checking significance correlations. Under the hypothesis that lL-lRA genotype may be associated with the risk of cervix cancer, we tested combination of lL-lRA genotype in models for squamous cell carcinoma (SCC), adenocarcinoma (AC), and stages of cancer (stages IA, IIA, IIIA, IB, IIB, IIB, IV); and the interaction of this gene with HPV 16 and HPV 18 were tested. The association between polymorphism in lL-lRA gene with the risk of cervical cancer was estimated by computing odds ratio (OR) and 95% confidence intervals (95% CI) by using a multivariate logistic regression analysis that included several potential confounding variables. Statistical analysis was performed using SPSS, version 10.0 (SPSS, Chicago, IL, USA) Epical version 3.2.

Table 2 Demographic characteristics of cervix cancer cases and controls
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Result

We have analyzed the genotype of the lL-lRA gene polymorphism in cervical cases and healthy controls derived from North Indian population.

Demographic variables for cases and controls are summarized in Table 2. Variables have also been categorized for squamous cell carcinoma (SCC) and adenocarcinoma (AC) cervix cancer. One hundred and twenty-nine were identified as SCC and 19 as AC. The average age in years was calculated as 48.55 ± 9.43 for cases and 48.81 ± 9.64 for controls. Compared to the controls, study group had age at the time of the marriage (16.36 ± 3.39; young), the birth of first child (18.39 ± 3.39) , and a greater mean number of children (4.11). Age at menarche and menopause were found to be comparable between cases and controls. Alleles frequencies lL-lRA are shown in Table 3.The frequency of allele A and B in cases is greater than control group. The association between the lL-lRA gene and cervical cancer is summarized in Table 4. The combination of CB and CA genotypes increased the risk of cervical cancer (OR = 2.24, 95% CI = 0.81–6.22). Table 5 shows interaction between lL-lRA and HPV type 16 and 18 in cervical cancer patients and healthy controls. There was a strong significantly protective association between heterozygous AB genotype and HPV 18 (OR = 0.11, 95% CI = 0.04–0.30, p = 0.0000000). Similarly this result was demonstrated, in combined AB + BB genotypes with HPV 18 (OR = 0.12, 95% CI=0.05–0.30, p = 0.000) and with HPV type 16 + 18 (OR = 0.18, 95% CI = 0.08–0.38, p = 0.000005).The association of lL-lRA genotype with type of cancer are briefed in Table 6. There was high protective significant association between heterozygous genotype AB with adenocarcinoma (OR = 0.19, 95% CI = 0.09–0.40, p = 0.0000002). The association between the lL-lRA genotype and the stages of cervical cancer are given in Table 7. We found a statistically highly significant protection between heterozygous AB genotype in stage IA (OR = 0.03, 95% CI = 0.00–0.21, p = 0.0000000), stage IIA (OR = 0.14, 95% CI = 0.06–0.34, p = 0.0000000), stage IIIA (OR = 0.38, 95% CI = 0.23–0.63, p  = 0.00008) stage IB (OR = 0.11, 95% CI = 0.04–0.30, p = 0.0000000), stage IIB (OR = 0.37, 95% CI = 0.20–69, p = 0.001); and AB + CB + BB genotypes in stage IA (OR = 0.14, 95% CI = 0.06–0.31, p = 0.000), stage IIIA (OR = 0.28, 95% CI = 0.15–0.53, p = 0.0000300), stage IIB (OR = 0.16, 95% CI = 0.08–34, p = 0.000).

Table 3 Allele frequency (%) of interleukin-1 receptor antagonist among women with cervical cancer and the healthy controls
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Table 4 Genotype IL-1RA gene in case and control
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Table 5 Assessments of interaction between IL-1RA and HPV in cervical cancer and control
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Table 6 Determination of interaction between IL-1RA genotype and type of cervical cancer
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Table 7 Association between the IL-1RA genotypes and stage of cervical cancer
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Discussion

Identification of SNPs (single nucleotide polymorphisms) in human genome has great implications in the study of disease susceptibility. The SNPs in lL-lRA gene (VNTR) have been found to be associated with different immunological diseases [3]. Experimental data supported the role for lL-lRA cytokine as autocrine or paracrine stimuli in murine and human carcinogenesis [34]. Decades of studies have confirmed that cervical infection by high-risk HPV types is a precursor event to cervical cancer. The natural history of cervical cancer as a continuous single disease process progressing gradually from mild cervical intraepithelial neoplasia (CIN1), to more severe neoplasia and micro-invasive lesion (CIN2 or CIN3) and finally to invasive disease has been the basis for diagnosis, therapeutic measure, and secondary preventive strategies [16]. The plasma level of IL-RA was influenced by the number of repeats in the IL-RA VNTR [1] and the allele with two repeats (designated as allele B in this study) has been reported to be associated with various diseases like, vestibulite, ulcerative colitis, alopecia areala, psoriasis, autoimmune conditions [2, 19], and idiopathic recurrent miscarriage [31]. El-Omar et al. [12] and Machado et al. [20] have reported an association of the IL-1 gene cluster polymorphism with enhanced production of IL-1b and gastric cancer, respectively. Their finding complements the most widely accepted multistage model of gastric carcinogenesis and underlines the fact that host genetic factors may determine why some people infected with Helicobacter pylori develop gastric cancer while others do not [12, 20]. This hypothesis can be transferred and applied to other solid tumor such as cervix cancer. Sehouli et al. [26] established that lL-lRA associated with cervical cancer and Allele 2 heterozygous has a greater risk in developing cervical cancer (p = 0.0400000). Mustea et al. [22] reported that allele 2 of the gene encoding for IL1-RA as a genetic determinant of cervical cancer. Our study confirmed these data (p = 0.0000200) in patient who were suffering from adenocarcinoma. The allele frequencies were different between cases and controls. Therefore, we did not find any controversy with pervious studies in North Indian population [3]. The polymorphism of IL-1RA seems to be involved in the induction of different solid tumors [26]. Bid et al. [4] suggested that IL-1RA intron 2 polymorphism plays a prominent role in bladder cancer in North India population. Grimm et al. [15] also demonstrated the same result with respect to vulvar carcinogenesis. Viet et al. [32] found that the IL-1RA polymorphism is associated with colorectal carcinogenesis. Hu et al. [17] showed that the allele 2 of IL-1RN (IL-RN×2) increased the risk of lung cancer in the Chinese population. Glas et al. [14] revealed that the homozygous genotype IL-1RN×2/2 of the IL-RN gene is strongly associated with early-stage gastric cancer (p < 0.0001)

So, lL-lRA mutation serum provided information about the prognosis of the patients. We observed association between specific alleles and clinical feature such as stage, and histological type in cervical cancer patient. Regarding other cancers such as ovarian cancer, no relation was found between specific allele and clinical feature [25]. Also, we demonstrated the influence of heterozygous AB genotype and AB + BB genotype IL-RA on risk of cervical cancer in patients showed high-risk of HPV 16 and 18 types. Our study showed that heterozygous AB genotype lL-lRA was increased in cervical cancer patients who were prone to high-risk of types 16 and 18. This is perhaps the first study until now that provided data regarding interaction between lL-lRA and HPV in cervical cancer and the first report on polymorphic changes in gene encoding IL-RA in patient with cervical cancer from North Indian population. The study concluded that cervical cancer is known to be a polygenic disease and genetic factors play an additional role in the indication of its malignancy, whereas only some of the genes were identified in cervical cancer patients. Some of them can mask potential influences of polymorphisms in the lL-lRA gene. These must be included in a panel of polymorphic genes which are associated with cervical cancer, similar to the gene regulating cell cycle [7] and MTHFR [29].