|Ahead of print
Association of p73 G4C14-A4T14 and p53 codon 72 polymorphism with cervical cancer in Chinese population
Haiyan Guo1, Zhang Wen2, Shaodi Yang3, Hongyan Qi1
1 Department of Obstetrics and Gynecology, Xi'an No. 4 Hospital, Xi'an, Shaanxi 710004, China
2 Shenzhen Bao'an Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518133, China
3 School of Automation, central South University, changsha, Hunan 410083, China
|Date of Submission||13-Jun-2019|
|Date of Decision||01-Sep-2019|
|Date of Acceptance||02-Oct-2019|
|Date of Web Publication||02-Jul-2021|
Department of Obstetrics and Gynecology, Xi'an No. 4 Hospital, Xi'an, Shaanxi 710004
Source of Support: None, Conflict of Interest: None
Background: Cervical cancer is known to be the fourth most common cancer among women globally. In various factors, genetic factors have been considered as one major risk factor for cervical cancer. The research of genetic susceptibility to cervical cancer can be greatly helpful in studying the complex mechanism. This study was conducted to identify whether polymorphic variants of p73 G4C14-A4T14 and tumor protein p53 (p53) codon 72, either independently or jointly, might be associated with the risk of cervical cancer.
Methods: The genotypes of p73 G4C14-A4T14 and p53 codon 72 polymorphisms of peripheral blood DNA from 190 cervical cancer patients and 210 controls were investigated using polymerase chain reaction with confronting two-pair primers and polymerase chain reaction-restriction fragment length polymorphism, respectively.
Results: The frequency of p73 G4C14-A4T14 AT/AT (P = 0.013) or p53 codon 72 GG (P = 0.026) genotype was associated with an increased risk of cervical cancer by comparing with the p73 G4C14-A4T14 GC/GC or p53 codon 72 CC genotype, respectively. In addition, the interaction between the p73 G4C14-A4T14 and p53 codon 72 polymorphisms increased the risk of cervical cancer in a multiply manner, with the odds ratio being 3.692 (95% confidence interval =2.106-6.473) for subjects carrying both p73 G4C14-A4T14 GC/AT+AT/AT and p53 codon 72 GG genotypes.
Conclusion: These results suggest that there is a statistical difference between p73 and p53 gene polymorphism and the risk of cervical cancer in Chinese women, and there is a potential gene-gene interaction in the incidence of cervical cancer.
Keywords: Cervical cancer, p53, p73, polymorphism, susceptibilityKey Message The p73 gene G4C14-A4T14 and p53 gene codon 72 polymorphisms are independently and jointly associated with the risk of cervical cancer in the Chinese population.
| » Introduction|| |
cervical cancer is the fourth most common cancer in women worldwide.,, The incidence of cervical cancer in underdeveloped countries is significantly higher than that in developed countries, especially in China. Human papilloma virus (HPV) is reported as a main reason for development of cervical cancer, with 99.7% cases were infected in the worldwide investigation. Except for HPV infection, lack of prophylactic vaccinations or cancer screening, immune system deficiency, herpes, smoking, age, sexual behavior and genetic factors are identified in epidemiologic studies as other major risk factors for cervical cancer. HPV genome has six early genes (E1, E2, E4-E7), and E6 can immortalize the keratinocytes as an oncoprotein, which can interacts with tumor suppressor gene p53 to prevent apoptosis. Thus, genetic susceptibility may contribute to cervical cancer development in a complex mechanism.
p73 gene is located on chromosome 1q36-33. It is the first examined homologue of tumor suppressor gene p53, and the structure is similar to p53. When p73 is overproduced, p73 may induce apoptosis to activate the p53 responsive genes transcription and inhibit cell growth in a p53-like manner. p53 gene is located on the short arm of chromosome 17p13.1. It is a regulatory factor in the cell growth cycle, and it is associated with such important biological functions as cell cycle regulation, DNA repair, cell differentiation and apoptosis., Whether the biological functions of p73 and p53 gene independently or whether their gene family relationship binds them in some pathway of cell proliferation and oncogenesis is unclear.
Single nucleotide polymorphisms (SNPs) mainly refer to the DNA sequence polymorphisms caused by the variation of single nucleotides at the genome level. It is the most common type of genetic variation, accounting for more than 80% of all known polymorphisms. Two SNPs of guanine-to-adenine (G-A) and cytosine-to-thymine (C-T) respectively at 4th and 14th position of the second exon p73 gene named G4C14-A4T14 (rs2273953, rs1801173). They are linked naturally as double nucleotide polymorphisms, which can replace each other. In functional aspect, it exists in transcriptional regions capable of forming a stem-ring structure and affects gene expression. Reports showed that p73 G4C14-A4T14 SNP was associated with the risk of several cancers. A polymorphism at codon 72 in exon 4 of the tumor suppressor gene p53, results in translation to either arginine (GG; Arg) or proline (CC; Pro), and the variant lead to an amino acid change in the protein product. p53 codon 72 polymorphism was also reported to be associated with the risk of various malignancies.,,
This present study is aimed at investigating a case–control study to identify the association between p73 G4C14-A4T14 and/or p53 codon 72 polymorphism(s), and cervical cancer risk among Chinese people.
| » Materials and Methods|| |
Study subjects and samples
We recruited 190 newly diagnosed patients with cervical cancer and 210 cancer-free controls, including 171 adenocarcinoma, 15 squamous cell and 4 others histology types. Patients were recruited between January 2014 and June 2016 at Xi'an No. 4 Hospital (Xi'an, China). Final diagnoses of cases were confirmed by routine histopathological examination. The selection of criteria for the controls included no individual history of cancer and frequency-matched by age. All subjects were unrelated ethnic Han Chinese. At recruitment, written informed consents about the study were obtained from all the patients and controls. Each participant was interviewed to collect information on demographic characteristics. The research protocol was approved by the Institutional Review Board of the hospital.
Genomic DNA was extracted from ethylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood samples by phenol-chloroform extraction., DNA was stored at -80°C until use. Main reagents were as follows: KI (potassium iodide), 0.9% NaCl (sodium chloride), chloroform/isoamyl alcohol (24:1), isopropanol, 70% ethanol, and all reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Genotyping of p73 G4C14-A4T14 polymorphism
The p73 G4C14-A4T14 genotypes were detected using polymerase chain reaction with confronting two-pair primers (PCR-CTPP) technique as previously described by our study. The PCR assay was performed in GeneAmp* PCR System 9700 (Applied Biosystems, America). The PCR profile consisted of an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 40 seconds, 60°C for 40 seconds, and 72°C for 40 seconds, and a final extension at 72°C for 10 minutes. PCR was done in a volume of 15 μL reaction using four primers: F1: 5'-CCA CGG ATG GGT CTG ATC C-3'; R1: 5'-GGC CTC CAA GGG CAG CTT-3'; F2: 5'-CCT TCC TTC CTG CAG AGC G-3'; R2: 5'-TTA GCC CAG CGA AGG TGG-3'. The fragments were visualized in 2% agarose gel electrophoresis. AT/AT allele produce two bands of 428 bp and 270 bp; GC/GC alleles produce two bands of 428 and 193 bp. Thus, the GC/AT heterozygote produce three bands, corresponding to 428, 270, 193 bp. To test the reliability of genotyping by PCR-CTPP, 50 randomly selected samples were re-tested by DNA sequencing. The results of PCR-CTPP genotyping and sequencing analysis were completely consistent [Figure 1].
|Figure 1: (a) The PCR-CTPP analyses of p73 G4C14-A4T14 polymorphism. M = 100bp marker; Lane 1, 5: GC/GC genotype (428, 193 bp); Lane 2, 6: GC/AT genotype (428, 270, 193 bp); Lane 3, 4; AT/AT genotype (428, 270 bp). (b) The direct-sequence analysis for genotypes of p73 G4C14-A4T14 polymorphism (GC/GC genotype; GC/AT genotype; AT/AT genotype)|
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Genotyping of p53 codon 72 polymorphism
p53 codon 72 genotypes were detected using PCR-restriction fragment length polymorphism (RFLP) technique, and the PCR primers were as follows: 5' -TCC CCC TTG CCG TCC CAA-3' and 5' -CGT GCA AGT CAC AGA CTT-3' (Sangon Biotech, Shanghai, China). The PCR assay was performed in GeneAmp* PCR System 9700 (Applied Biosystems, America). The PCR profile consisted of an initial denaturation at 94°C for 2 minutes; followed by 35 step cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes; and a final extension step of 72°C for 10 minutes. Digestion was done in 20 microliters reaction volume using BstUI restriction enzyme (New England Biolabs, Britain) for 2 hours at 60°C according to the instructions of the manufacturer. The fragments were visualized in 2.5% agarose gel electrophoresis. C alleles produced a single band of 279 bp, which was caused by lack of BstUI restriction site; G alleles were digested to produce two bands of 119 and 160bp. Thus, the CG heterozygote produced three bands, corresponding to 279, 119, 160 bp. To test the reliability of genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 50 randomly selected samples were re-tested DNA sequencing. The results of PCR-RFLP genotyping and sequencing analysis were completely consistent [Figure 2].
|Figure 2: (a) The PCR-CTPP analyses of p53 codon 72 polymorphisms. M = 100bp marker; Lanes 1, 3, 5, 6, 7: GG homozygous genotypes (160, 119 bp); Lanes 2, 4: CG heterozygous genotype (279, 160, 119 bp); Lane 8: CC homozygous genotypes (279 bp). (b) The direct-sequence analysis for genotypes of p53 codon 72 polymorphisms (CC genotype; CG genotype; GG genotype)|
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The Statistical Package for the Social Sciences, version 17.0 (SPSS, Inc.) was used for all statistical analyses. We used the χ2-test to evaluate the clinicopathological characteristics between cases and controls. The association between the polymorphisms and cervical cancer risk were determined by odds ratio (OR) and 95% confidence interval (CI) by unconditional logistic regression models. P < 0.05 was considered statistically significant.
| » Results|| |
Characteristics of study subjects
The frequency distribution of selected characteristics of the cases and controls is showed in [Table 1]. The frequency distribution between age, alcohol consumption, and family history had no significant difference in cases and controls (P > 0.05). However, there was a statistical difference regarding smoking, which the cigarette smokers' frequency among cases and controls was 17.9% and 6.2%, respectively, (χ2 = 13.178, P = 0.000).
|Table 1: Demographic characteristic of cervical cancer cases and controls|
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Distribution of p73 G4C14-A4T14 and p53 codon 72 polymorphisms
The allele and genotype frequencies of p73 G4C14-A4T14 and p53 codon 72 polymorphisms are presented in [Table 2]. The observed genotype frequencies of p73 G4C14-A4T14 and p53 codon 72 polymorphisms in controls did not deviate significantly from those expected from the Hardy–Weinberg equilibrium. The frequencies of p73 G4C14-A4T14 AT allele and p53 codon 72 G allele were 28.9 and 65.5% among cases and 20.0 and 54.0% among controls, respectively. According to [Table 2], only AT/AT genotype is statistically different, with the AT/AT genotype being more frequent among cases compared with controls (28.9% versus 20.0%; P < 0.05). Similarly, according to [Table 2], only GG genotype is statistically different, with the GG variant being more frequent among cases compared with controls (52.6% versus 30.9%; P < 0.05).
|Table 2: Genotype and allele frequencies of p73 and p53 among cases and controls and their association with the risk of cervical cancer|
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Association between p73 G4C14-A4T14 and p53 codon 72 polymorphisms with cervical cancer risk
An unconditional logistic regression model was used to estimate the association between genotypes and the risk of cervical cancer [Table 2]. The p73 G4C14-A4T14 AT/AT genotype was associated with an increased risk for the development of cervical cancer (χ2 = 6.118, P = 0.013; OR = 2.381, 95%CI = 1.181–4.802), compared with the GC/GC genotype. Likewise, the p53 codon 72 GG genotype was also associated with an increased risk of developing cervical cancer (χ2 = 4.948, P = 0.026; OR = 1.801, 95%CI = 1.070–3.032), compared with the CC genotype. However, the heterozygous genotypes for both polymorphisms (p73 G4C14-A4T14 GC/AT or p53 codon 72 CG) were not associated with the risk of cervical cancer (P > 0.05). The p73 G4C14-A4T14 GC/AT and AT/AT or p53 codon 72 CC and CG genotypes were combined into one group for subsequent analysis. We examined whether there was a statistical joint effect between the p73 G4C14-A4T14 and p53 codon 72 polymorphisms in [Table 3]. The observation showed that patients carrying the p73 G4C14-A4T14 Gc/AT +AT/AT genotype were more likely to carry the p53 codon 72 GG genotype than the controls (31.5% versus 11.9%; χ2 = 21.836, P < 0.05). Moreover, the OR increased to 3.692 (95% CI = 2.106–6.473) among subjects carrying both p73 G4C14-A4T14 GC/AT+AT/AT and p53 codon 72 GG genotypes.
|Table 3: Risk of cervical cancer associated with p73 genotypes by p53 genotypes|
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| » Discussion|| |
To the best of our knowledge, it is the first time to jointly investigate the association between the p73 G4C14-A4T14 and the p53 codon 72 polymorphisms with cervical cancer risk in Chinese Han population. In this hospital-based case–control analysis among Chinese Han population, we demonstrated that both p73 G4C14-A4T14 and p53 codon 72 polymorphisms were associated with an increased risk for development of cervical cancer. Furthermore, a multiplicative interaction between p73 G4C14-A4T14 GC/AT+AT/AT and p53 codon 72 GG genotypes was also detected.
p73 gene is a related gene that belongs to tumor suppressor gene p53 superfamily, and it was discovered by Kaghad et al. in 1997. The p73 G4C14-A4T14 is a potentially functional polymorphism, which can form a stem-loop structure and control p73 gene expression. It plays an important role in tumorigenesis and affects susceptibility of different cancers. It has a few reports about the relationship between p73 G4C14-A4T14 polymorphism and the cervical cancer susceptibility, currently. In southwestern European population, craveiro et al. indicated that p73 AT allele carriers have two-fold increased susceptibility to the formation of high grade squamous intraepithelial lesion in cervical cancer by comparing with other genotypes. According to Niwa et al. study conducted among Japanese population, there is no significant difference in the p73 G4C14-A4T14 genotype frequency between cases and controls (OR = 1.57; 95%CI = 0.99–2.48, P = 0.053). In Chinese Xinjiang Uigur population, Zheng et al. investigated that p73 G4C14-A4T14 polymorphism may not be associated with cervical cancer by PCR-RFLP and PCR-CTPP. Our findings confirmed the results of craveiro et al., an association between p73 G4C14-A4T14 AT/AT genotype and an increased risk for the formation of cervical cancer in Chinese Han population.
The mutation or deletion of p53 gene is related to more than 50% of cancers. Protein encoded by p53 gene is a transcriptional factor that controls the start of the cell cycle. The p53 gene was defined as “the guardian of the genome”; many literatures had reported on the association between p53 condon 72 polymorphism and the cervical cancer, but the conclusions were inconsistent. For instance, Pillai et al. and Bansal et al. found no evidence for any association between p53 codon 72 polymorphism and cervical cancer patients in Indian population., However, Nagpal et al. showed a significant difference in p53 codon 72 Arg/Arg homozygous allele in high-risk HPV-infected cervical cancer cases and HPV-negative cancer in Indian women. Sousa et al. Collected 27 studies published within 12 different European populations between 1998 and 2005 and concluded that p53 codon 72 Arg/Arg genotype may not be a representation for a cervical cancer risk marker in majority of the European countries. However, some European countries with low incidence rates, p53 codon 72 polymorphism was associated with cervical cancer risk. The meta-analysis of Zhou et al. assessed 28 case–control studies among Asians and showed p53 codon 72 polymorphism may be a risk factor. However, no associations were found among China, Japan and Korea populations. Consistent with that of Nagpal et al., our study reveals that the p53 codon 72 GG genotype was associated with an increasing risk for cervical cancer when compared with the p53 codon 72 CC genotype.
We investigated the joint association between p73 and p53 polymorphisms in the incidence of cervical cancer. p73 gene has high homology with p53 gene on its structure and function. When p53 gene is inactivated, the overexpression of p73 can activate the transcription of p53 target gene, induce apoptosis, inhibit cell growth, and even induce tumorigenesis. The only two relevant literature reported no association between p73 G4C14-A4T14 and p53 codon 72 polymorphisms are those of Niwa et al. and Jha et al. among Japanese and north Indian population, respectively., Therefore, the association between p73 G4C14-A4T14 and p53 codon 72 polymorphisms required further study with more samples in different racial and ethnic groups.
| » conclusion|| |
Our case–control study showed the association between the p73 G4C14-A4T14 and/or p53 codon 72 polymorphisms and an increased risk of developing cervical cancer among Chinese women. Further, the association of p73 G4C14-A4T14 and p53 codon 72 polymorphisms with the risk of cervical cancer displayed a multiplicative gene–gene interaction. If confirmed by other studies, the underlying mechanisms of genetic variations in p73 gene G4C14-to-A4T14 and p53 codon 72 polymorphisms as presented here may be valuable for the cervical cancer therapy and diagnosis.
This research is supported by the Technology Planning Project of Shaanxi Province (grant no. 2018SF-147).
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]