|Year : 2017 | Volume
| Issue : 1 | Page : 23-27
Analysis of prostaglandin-endoperoxide synthase-2 gene polymorphisms and risk of cervical cancer in an East Indian population: A case–control study
Dipanshu Sur, Ratnabali Chakravorty
MAGS Medical and Research Center, Kolkata, West Bengal, India
|Date of Web Publication||3-Jul-2017|
MAGS Medical and Research Center, Kolkata - 700 091, West Bengal
Source of Support: None, Conflict of Interest: None
Background: The prostaglandin-endoperoxide synthase-2 (PTGS-2) gene appears to play a role in inflammation or tumor and mitogenesis. Genetic polymorphisms in PTGS-2 might contribute to differential PTGS-2 expression and subsequent interindividual variability in susceptibility to cancer.
Aim: The goal of this study is to identify genetic variants of PTGS-2 gene in women of East India, which may associate with risk of cervical cancer.
Materials and Methods: We enrolled 200 histopathologically confirmed patients with cervical cancer (age 18–60 years) (cases) and their corresponding sex-matched 200 normal individuals (controls). To identify genetic variants responsible for cervical cancer, we performed sequence analysis of PTGS-2 genes. Questionnaire survey was conducted to comprehend the demographic data, smoking status, and cancer stage of patients.
Results: The genotype frequency of rs689466 polymorphism was significantly different between case and control groups (P < 0.001). Compared with the wild-type genotype AA, the variant genotype GG was associated with 20-fold increased risk (P < 0.001; odds ratio = 20.76; 95% confidence interval [CI]: 2.86–160.73) for cancer patients. The rs5275: exon1-+837T>C polymorphism was not associated with cancer risk although this allele was correlated with decreased risk (P = 0.701; odds ratio = 0.71; 95% CI: 0.26–1.90). CC genotype was more frequently found in controls as compared with cases and showed an inverse association with the development of cervical cancer, thus suggesting a possible protective effect.
Conclusions: PTGS-2 genotype rs689466:—1195A/G gene polymorphism demonstrated strongly associated with cervical cancer disease. However, exon1-+837T > C polymorphism was not associated with cancer risk in East Indian women. Further studies evaluating the role of PTGS-2 gene polymorphisms in ethnically diverse populations and a larger cohort may help in understanding the etiopathogenesis of cervical cancer in women worldwide.
Keywords: Cervical cancer, East India, polymorphism, prostaglandin-endoperoxide synthase-2, susceptibility
|How to cite this article:|
Sur D, Chakravorty R. Analysis of prostaglandin-endoperoxide synthase-2 gene polymorphisms and risk of cervical cancer in an East Indian population: A case–control study. Asian J Oncol 2017;3:23-7
|How to cite this URL:|
Sur D, Chakravorty R. Analysis of prostaglandin-endoperoxide synthase-2 gene polymorphisms and risk of cervical cancer in an East Indian population: A case–control study. Asian J Oncol [serial online] 2017 [cited 2018 Jul 16];3:23-7. Available from: http://www.asjo.in/text.asp?2017/3/1/23/209342
| Introduction|| |
In worldwide, the second most common cancer in women is cervical cancer., The high-risk human papillomaviruses (HPVs) (e.g., HPV-16 and HPV-18) are a major source of cervical cancer in women. However, the underlying cellular and molecular mechanisms in the pathogenesis of cervical cancer remain largely unexplored. Genetic factors play important roles in initiation and progression of diverse diseases. Prostaglandin-endoperoxide synthase-2 (PTGS-2) has been implicated in cervical carcinogenesis.
Cyclooxygenases (COXs), also known as PTGS, are rate-limiting enzymes that convert free arachidonic acid into several prostaglandins, namely, prostaglandin E2 (PGE2), inducing the immune response through the “inflammation pathway” [Figure 1].
PTGS-2 is constitutively expressed in some tissues in physiological conditions, such as the endothelium, kidney, and brain and in pathological conditions, such as cancer. PTGS-2 is responsible for production of inflammatory prostaglandins. Upregulation of PTGS-2 is also associated with increased cell adhesion, phenotypic changes, resistance to apoptosis, and tumor angiogenesis. In cancer cells, PTGS-2 is a key step in the production of PGE2, which plays important roles in modulating motility, proliferation, and resistance to apoptosis. Recent studies have demonstrated that genitourinary carcinomas, including cancer of the uterine cervix, may be regulated by PTGS activity.,,
COX-2 is an enzyme encoded by the PTGS2 gene, which spans about 8.3 kb on chromosome 1q25.2-q25.3 and has 10 exons and 9 introns [Figure 2].
|Figure 2: Human prostaglandin-endoperoxide synthase-2 gene (cytogenetic band: 1q25.2-q25.3)|
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PTGS-2 +8473T/C (rs5275) is located at the 3'-untranslated region and could alter gene expression through both messenger RNA stability and translational efficiency in vitro. The −765G/C polymorphism (rs20417) is located within a putative SP1-binding site that lowers promoter activity, thereby resulting in downregulation of COX-2 expression. As PTGS-2 is emerging as an important candidate gene in carcinogenesis, the present study was designed to evaluate the association of PTGS-2 +8473T/C and −765G/C gene polymorphisms with risk of cervical cancer. The biological location of these single-nucleotide polymorphisms (SNPs) advocates the importance of these variations, which may confer a modest effect on the gene product. The −1195A>G polymorphism is located in the promoter region of PTGS-2, characterized by an adenine (A) to guanine (G) transition at position −1195 from exon1. So far, functional studies have shown that the −1195A>G polymorphism regulates PTGS-2 transcription activity and COX-2 expression, by creating different recognition-binding sites for nuclear proteins.
To clarify the hypothesis that SNP variants of COX-2 are associated with the risk of cervical cancer, we analyzed the genetic polymorphisms of three PTGS-2 SNPs, namely, −1195A/G (rs689466), −765G/C (rs20417), and +8473T/C (rs5275), in a large eastern part of cervical cancer population (control/case = 200/200).
| Materials and Methods|| |
We have designed a retrospective case–control study gathering patients with cervical cancer (200) and healthy controls (200).
This study included 400 participants: 200 histologically confirmed cervical cancer patients and 200 cancer-free controls, from the eastern region of India and recruited at the Obstetrics and Gynecology Outpatient Department of MAGS Medical and Research Center, West Bengal, Kolkata. Written informed consent was obtained from all recruited participants before their inclusion in the study, according to the Declaration of Helsinki. This research project was approved by the Ethics Committee (ref. 14/A; 667).
Sample collection and biological processing
Blood samples were collected from histopathologically confirmed patients of cervical cancer (Stages I, II, III, and IV) and age-matched, similar ethnicity, unrelated female controls free from cervical cancer, human immunodeficiency virus, and with no family history of malignancy, allergy, diabetes, or cardiovascular disease. Our control group did not have any chronic clinical problem and/or disease manifestation. Clinical diagnosis of cervical cancer patients was performed by trained medical personnel. Approximately 5 ml of peripheral blood samples was collected from the participants with the help of the collaborating doctors. Ethylenediaminetetraacetic acid (EDTA) was used as an anticoagulant. Genomic DNA extracted from fresh whole blood by QIAamp Blood Kit (Qiagen, Hilden, Germany). Genomic DNA used as a template in the polymerase chain reaction (PCR).
Polymerase chain reaction
PCR was carried out in a total reaction volume of 30 μl containing 50–100 ng genomic DNA, standard buffer, MgCl2(as appropriate), 0.2 mM of each dNTP, 0.5 μM of each primer [Table 1], and 0.8 units of TaqDNA polymerase (Invitrogen, Carlsbad, CA, USA) in a Veriti™ 96-well thermocycler (Applied Biosystems, Foster City, CA, USA). PCR conditions used were as follows: initial denaturation of 95°C for 5 min, 35 cycles at 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s, followed by the final extension at 72°C for 5 min. The PCR products were analyzed by electrophoresis in 1.5% agarose gels and visualized under ultraviolet light. Only those PCR products that had a single amplification product with no evidence of nonspecific amplification were used for DNA sequencing. The PCR products free of contaminating bands due to nonspecific amplification were column purified using a Qiagen PCR purification kit (Qiagen, Hilden, Germany), and bidirectional sequencing was performed in an ABI PRISM 3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA) using dye-termination chemistry. The sequences were analyzed using pairwise BLAST to examine if there was any change from the normal sequence available in the database. The PTGS-2 polymorphisms included in this study (rs20417, rs689466, and rs5275) were selected based on: (1) previous evidence of association with cervical tumor risk, (2) biological plausibility, and (3) minor allele frequency of at least 15%.
|Table 1: Primer sequences used for the analysis of human prostaglandin-endoperoxide synthase-2|
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Allelic and genotypic associations of each SNP were tested using Chi-squared or Fisher's exact test where appropriate. Hardy–Weinberg equilibrium of each SNP in the case and control individuals was also examined using a Chi-squared test. To calculate any statistically significant difference of continuous independent variables such as age, within the control and patient groups, we used Student's t-test. Mann–Whitney U-test was used to analyze nonparametric variables. All tests were done using GraphPad InStat software (GraphPad Software Inc., San Diego, CA, USA). The odds ratio and 95% confidential intervals were also calculated using the same software.
Power was estimated using genetic power calculator. The sample size was calculated using QUANTO software, version 1.2.4, May 2009. Continuous results are expressed as mean ± standard deviation.
Anthropometric and clinical characteristics
Cervical cancer patients and control participants were comparable in age (48.27 T 10.04 vs. 46.95 T 9.81 years). Stages I–IV were diagnosed in 200 cervical cancer patients (Stage I, n = 21 [10.5%]; Stage II, n = 71 [35.5%]; Stage III, n = 104 [52.0%]; and Stage IV, n = 4 [2.0%]). Clinical diagnosis/staging of cervical cancer cases was performed as per the guidelines of the International Federation of Gynecology and Obstetrics. Majority of cervical cancer patients had Stage III. Tobacco users, either smokers or chewers, constituted 27.5% of the patients.
Genes polymorphism and cervical cancer
Genotype and allele frequency distribution of PTGS-2 (+8473T/C; rs5275), PTGS-2 (−1195A/G; rs689466), and PTGS-2 (−765G/C; rs20417) gene polymorphisms between cervical cancer cases and controls is depicted in [Table 2] and [Table 3]. In control participants, the genotype frequencies for both polymorphisms were in Hardy–Weinberg equilibrium. Significant association was observed in PTGS-2 −765G/C and PTGS-2 −1195A/G gene polymorphism (P ≤ 0.001; OR, 3.06; 95% confidence interval [CI], 1.94–4.82 and P ≤ 0.001; OR, 14.87; 95% CI, 1.94–114.26). However, no significant association was observed in PTGS-2 +8473T/C gene.
|Table 2: Genotype distribution of prostaglandin-endoperoxide synthase-2 (-1195A/G; rs689466), prostaglandin-endoperoxide synthase-2 (+8473T/C; rs5275), and prostaglandin-endoperoxide synthase-2 (-765G/C; rs20417) gene polymorphisms in cervical cancer cases and controls|
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|Table 3: Allele distribution of prostaglandin-endoperoxide synthase-2 gene polymorphisms in the study|
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Prostaglandin-endoperoxide synthase-2 (+8473T/C and J765G/C) gene polymorphisms and clinical stages of cervical cancer
Stages I–IV were diagnosed in 200 cervical cancer patients (Stage I, n = 21 [10.5%]; Stage II, n = 71 [35.5%]; Stage III, n = 104 [52.0%]; and Stage IV, n = 4 [2.0%]). To investigate any possible association between PTGS-2 (+8473T/C, −1195A/G, and −765G/C) gene polymorphisms and clinical stages of cervical cancer, we performed a case-only analysis; however, our findings did not reveal any significant association with PTGS-2 gene polymorphisms and clinical Stage I, II, III, or IV of cervical cancer. COX-2 (+8473T/C and −765G/C) gene polymorphisms and tobacco usage among cervical cancer patients in the present study comprising 200 cervical cancer patients, 55 (27.5%) were tobacco users and 145 (72.5%) were nonusers. The majority of women with tobacco usage had a chewing habit but even a small percent of smokers in the study reported considerable chewing of tobacco. A case-only study analysis did not demonstrate any significant association between PTGS-2 (+8473T/C, −1195A/G, and −765G/C) gene polymorphisms and modulation of cervical cancer risk due to tobacco usage.
| Discussion|| |
PTGS-2 is emerging as an important candidate gene in carcinogenesis, including cancer of the uterine cervix. To date, the effect of SNP of PTGS-2 on cervical cancer susceptibility has not been evaluated in the East Indian population, especially in West Bengal subpopulation. However, the precise immunobiological mechanisms associated with cervical cancer susceptibility in women are not yet fully elucidated. The present study aimed to investigate the role of PTGS-2 gene polymorphisms with the risk of developing cervical cancer in East Indian women. Our results demonstrated that the significant association was observed in PTGS-2 −765G/C and PTGS-2 −1195A/G gene polymorphism (P ≤ 0.001; OR, 3.06; 95% CI, 1.94–4.82 and P ≤ 0.001; OR, 14.87; 95% CI, 1.94–114.26). However, no significant association was observed in PTGS-2 +8473T/C gene.
We did not find any significant association between clinical stages of cervical cancer and tobacco usage. To the best of our knowledge, this is the first study to explore the role of PTGS-2 (+8473T/C, −1195A/G, and −765G/C) gene polymorphisms in cervical cancer susceptibility in East Indian women.
The polymorphism has been reported to alter gene expression through both messenger RNA stability and translational efficiency. Lee et al. have previously investigated the role of PTGS-2 gene polymorphisms with risk of cervical cancer in Korean women; however, their study did not reveal any significant association between PTGS-2 +8473T/C gene polymorphism and cervical cancer susceptibility. Significant association was observed in PTGS-2 −765G/C and PTGS-2 −1195A/G gene polymorphism (P ≤ 0.001; OR, 3.06; 95% CI, 1.94–4.82 and P ≤ 0.001; OR, 14.87; 95% CI, 1.94–114.26). However, no significant association was observed in PTGS-2 +8473T/C gene. Subbaramaiah and Dannenberg  have indicated that PTGS-2 transcription is regulated by HPV-16 E6 and E7 oncoproteins by induction of a corepressor/coactivator exchange and is mediated by the activation of epidermal growth factor receptor-Ras-MAPK-AP1 pathway. A recent study by Kim et al. has implicated the involvement of nuclear factor-JB and activator protein-1 in PTGS-2 upregulation by HPV-16 E5 oncoprotein.
The role of promoter region polymorphism PTGS-2 −765G/C (rs20417) for cervical cancer susceptibility revealed a significant association in our study. Previous studies evaluating the association of PTGS-2 gene polymorphisms in cancer susceptibility in ethnically disparate populations worldwide have demonstrated inconsistent findings, thereby suggesting the relevance of host genetic factors in interindividual differences to disease susceptibility.
Both +8473T/C, −1195A/G, and −765G/C polymorphisms are located in the same gene, and we carried out disease risk at the haplotype interface. The complexity associated with the clinic-pathological events in cervical cancer progression is indeed interesting. We investigated any possible association between PTGS-2 (+8473T/C, −1195A/G, and −765G/C) gene polymorphisms and clinical stages of cervical cancer. Tobacco carcinogens have been associated with the risk of cervical malignancy. However, our data did not find any significant association between PTGS-2 (−1195A/G, +8473T/C, and −765G/C) gene polymorphisms and modulation of cervical cancer risk due to tobacco usage.
There are some limitations to the present study. First, we conducted a study with limited sample size and cervical cancer samples from a single cohort that might have influenced our findings. It is also plausible that limited study samples might lead to a relatively lower statistical power in certain subgroups, especially in case of tumor stage stratification. On the other hand, the strength of our study is that we have been able to explore the relationship between habitual substance use and the risk of cervical cancer from East India to provide us adequate information to well design the questionnaire and to adjust the influence from those potential confounding factors for the future studies. Future studies exploring PTGS-2 associations among diverse populations with sundry ethnicity may enhance our current understanding of the complex cellular and molecular mechanisms in cervical cancer susceptibility.
The authors acknowledge the study participants for giving their consent.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]