J Cancer 2019; 10(1):238-248. doi:10.7150/jca.27103

Research Paper

Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

Feng Ye1, Hanzhi Wang1, Jia Liu2, Qi Cheng1, Xiaojing Chen1, Huaizeng Chen1 Corresponding address

1. Women's Reproductive Health Key Laboratory of Zhejiang Province, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, P.R. China;
2. Department of Obstetrics and Gynecology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310006, P.R. China.

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How to cite this article:
Ye F, Wang H, Liu J, Cheng Q, Chen X, Chen H. Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population. J Cancer 2019; 10(1):238-248. doi:10.7150/jca.27103. Available from http://www.jcancer.org/v10p0238.htm

Abstract

Background and Aims: This study was aim to investigate the relationship between the four intron SNPs (rs3087404, rs2029167, rs2029166 and rs7296239) of SMUG1 and the susceptibility of cervical squamous cell carcinoma.

Methods: Four SMUG1 intron SNPs (rs3087404, rs2029167, rs2029166 and rs7296239) were genotyped by MA-PCR in 400 CSCCs, 400 CIN III and 1200 controls. qRT-PCR and Western blot were used to detect the SMUG1 mRNA and protein expression.

Results: Interestingly, we found that the homozygous GG of rs3087404 had a significantly increased risk of CIN III [OR=1.78(1.27-2.51), P= 0.001] and CSCCs [OR=4.04(2.94-5.55), P=0.000]. The individuals with G allele or G carrier (AG +GG) at rs3087404 were at higher risk for CSCCs [OR=1.34 (1.04-1.71), P= 0.022]. Similarly, the homozygous GG of rs2029167 also had an increased risk of CIN III [OR=2.56 (1.91-3.43), P= 0.000] and CSCCs [OR=4.05(3.02-5.44), P=0.000]. The individuals with G allele or G carrier (AG +GG) at rs2029167 were at higher risk for CINIII [OR=1.41(1.10-1.80), P= 0.006] and CSCCs [OR=1.91 (1.48-2.47), P= 0.000]. In HR-HPV positive group, both the homozygous GG of rs3087404 and the homozygous GG of rs2029167 had an increased risk to CIN III and CSCC. Stratified analysis of the number of sexual partners and the age of first sexual intercourse found that the rs3087404 (A/G) had a particularly high level of enrichment in the CIN III or CSCCs groups. About the rs2029167 (A/G), we only found a particularly high level of enrichment grouping by the number of sexual partners in the CIN III and CSCCs groups. Meanwhile, we also found that there is a correlation between the SNPs of SMUG1 rs3087404 (A/G) and rs2029167 (A/G) with tumor cell differentiation and family heredity. But we didn't find that there was an association between the deferent genotypes of SMUG1 rs2029166 and rs7296239 with SMUG1 gene mRNA or protein expression. During the linkage disequilibrium analysis between rs3087404 (A/G) and rs2029167 (A/G), the genotype with AA-GG [OR=3.14(1.95-5.05)], AG-GG [OR=2.45(1.58-3.89)], GG-AA [OR=2.24(1.28-3.90)] and GG-AG [OR=2.58(1.54-4.32)] significantly increased the risk of CIN III. More notably, this risk is much greater in CSCCs: AA-GG [OR=7.13(4.03-12.61)], AG-GG [OR=7.22(4.21-12.38)], GG-AA [OR=8.60(4.73-15.63)], GG-AG [OR=9.64(5.43-17.13)]. Additionally, most GG (rs3087404) genotypes were linkage GG-AG (44/77, 80/140) in the CIN III and CSCCs, while most GG (rs2029167) genotypes were linkage genotype AG-GG (79/145, 112/184) in the CIN III and CSCCs, respectively.

Conclusions: These findings suggested that there was association between the two genetic polymorphisms of SMUG1 rs3087404(A/G) and rs2029167(A/G) with the susceptibility of CIN III and CSCCs, and there was a linkage disequilibrium between the rs3087404 with the rs2029167 in CIN III and CSCCs. This particular linkage disequilibrium can be used as predictive biomarkers of CIN III and CSCC.

Keywords: SMUG1, intron, genetic variant, linkage disequilibrium, cervical squamous cell carcinoma, CIN III

Introduction

Around the world, cervical cancer (CC) is the fourth most common cancer among women, accounting for an estimated 529,572 diagnosed new cases and 274,967 deaths per year [1]. This cancer is the 3rd-leading cause of death in women' neoplasis worldwide and the morbidity of cervical cancer has increased recently [2]. In China, the cervical cancer has become the first major female cancer (98.9 per 100000) in addition to breast cancer. The mortality rate is up to 30.5 per 100000. And the incidence rate has the increasing trends [3].

Although several factors that contribute to cervical cancer development have been identified—mainly intrinsic factors (genetic), and extrinsic factors belonging to the high risk Human Papillomavirus (HR-HPV)—genetic factors show great potential as susceptibility or prognosis indicators [4,5]. Only a small fraction (~1%) of cervical HR-HPV infection outcomes to cervical neoplasia, and the factors determining risk of progression are not entirely understood [6]. Many genetic variants were associated with the risk of cervical cancer as supported by the epidemiological evidence [7].

Genomic instability due to DNA damage by carcinogens has been implicated in the initiation and development of cancer. DNA damage response and repair counteract the threats to genomic integrity, and variations in DNA repair capacity resulting from genetic variants could correlate with cancer predisposition [8-11]. The base excision repair (BER) pathway is the major DNA genetic damage repair pathway involved in genomic instability and tumorigenesis. Previous candidate gene studies showed that selected functional single nucleotide polymorphisms (SNP) in BER genes are associated with higher risk of several solid cancers [12-17].

Single-strand selective mono-functional uracil-DNA glycosylase (SMUG1) is one of the BER genes which remove uracil from double-stranded and single-stranded DNA to maintain genomic stability following oxidative attacks [18]. SNPs in this gene could have an effect on its enzyme capability of repairing DNA damage.

Xie et al evaluated the associations of 167 SNPs from 19 genes of the BER pathway with the risk of bladder cancer. 13 SNPs in 10 BER pathway genes were significantly associated with bladder cancer risk. The most significant SNP was rs2029167 in the SMUG1 gene [19]. Similar studies also found a correlation between SNP of SMUG1 with breast cancer [20] and colorectal cancer (CRC) [21]. Until now, there is no report of SNP of SMUG1 in cervical cancer.

In this large scale case-control study, the aim was to investigate the relationship between the four intron SNPs (rs3087404, rs2029167, rs2029166 and rs7296239) of SMUG1 and the susceptibility of cervical squamous cell carcinoma (CSCC). Genotyping analyses of the four SMUG1 SNPs were performed in 400 CSCCs, 400 precursor lesion CIN III and 1200 normal controls.

Materials and methods

Study samples selection

400 CSCC cases, 400 CIN III cases and 1200 normal controls were recruited from Zhejiang Province, China. The diagnosis was determined by two pathologists. All subjects were unrelated ethnic Chinese women and recruited between 2004 Jun to 2008 Dec. Normal controls were randomly selected from healthy women volunteers during gynecologic examinations. The inclusion criteria for healthy volunteers were without gynecological neoplasm, cytological findings, endometriosis, other solid cancer and immune disorders.

Patients with pathological diagnosis of CINIII and cervical squamous cell carcinoma (CSCC) were included in the study. Considering that patients with CINI and CINII have unstable disease progression, we excluded these patients and only selected patients with CINIII. All cases of CSCC were FIGO stage Ia-IIb, histologically confirmed primary cervical carcinoma, treated radical hysterectomy with pelvic lymph node dissection, and did not receive any anticancer therapy prior to their surgery. Patients who are eligible for any of the following criteria are excluded: over 70 years of age, with other serious complications, or previous malignant disease.

Of these, 201 CSCC patients, 357 CIN III patients and 609 normal controls agreed to provide cervical brush-off samples for detecting HR-HPV. This study was approved by the Medical Ethical Committee of Women's Hospital, School of Medicine, Zhejiang University (No.2004002). All patients signed informed consent.

DNA Extraction and Genotyping

Genomic DNA was extracted from anticoagulant peripheral blood using a DNA extraction kit according to the manufactor's guideline (Sangon Bioengineering Co., Shanghai, China). All DNA samples were dissolved in water and hypothermic preservation ready to use.

The four intron SNPs (rs3087404[A/G], rs2029167 [A/G], rs2029166 [C/T] and rs7296239 [C/T]) of SMUG1was detected by Modified polymerase chain reaction-mismatch amplification (MA-PCR) (As described in detail previously [22]). The PCR forward and reverse primers and product length were showed in following table 1.

 Table 1 

The PCR forward and reverse primers

SNP No.Forward primerReverse primerProduct length
rs3087404 [A/G]For “A5'-CTCATCAAGAGACTGCTGGA-3'5'-ACTTTCATTGTTCCATAACT-3'240bp
For “G5'-CTCATCAAGAGACTGCTGGG-3'
rs2029167 [A/G]For “A5'-GGGTGGTCCTCAGCTTGGCA-3'5'-GCAGTGACTGGCAGGAGGCG-3'184bp
For “G5'-GGGTGGTCCTCAGCTTGGCG-3'
rs2029166 [C/T]For “C5'-GCCATCTCTCATGGATTAAC-3'5'-TTATGAGATAGCAGTGACTG-3'228bp
For “T5'-GCCATCTCTCATGGATTAAT-3'
rs7296239 [C/T]For “C5'-CAGCCTCAACCCCAAAAGAC-3'5'-TGGCTAATGTTGAGCGAAAT-3'128bp
For “T5'-CAGCCTCAACCCCAAAAGAT-3'

The PCR was performed in a 25ul reaction mixture, containing 50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each dNTP and 1.0U of Taq DNA polymerase (TAKARA, Dalian, China). PCR undertook the following conditions: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30s, 57°C for 30s, and 72°C for 1min, and a final step of 72°C for 10min. The PCR products were developed by 1.5% agarose gel electrophoresis, stained with ethidium bromide and visualized with a TyphoonTM 9410 Imaging System (GE Healthcare, USA). All samples were tested twice in double blind by two different technicians, and the reproducibility of the experiment was 100%.

HR-HPV detection

HR-HPV infection was identified using the Hybrid Capture II(HC II) assay (Digene Diagnostics Inc., Gaitherburg, MD, USA) using probe B, which includes a pool of RNA probes for HR-HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Cervical sampling for HR-HPV DNA was performed with the Digene Cervical Sampler.

SMUG1 mRNA expression by qRT-PCR

Freshly frozen tumor tissues of eighty seven CSCCs were selected. Total RNA was extracted using Trizol reagent according to the manufacturer's protocol (Invitrogen, USA). Total RNA was treated with RNase-free DNase I. cDNA was reversed transcription and used as a template for qPCR detection. The following PCR primer pairs were used for quantitative amplification; 95°C 30s, 40 cycles at 95°C 5s followed with 60°C 30s. The primers of SMUG1 (mRNA: NM_001243787.1) were 5'-CGCAACTACGTGACTCGCTA-3'; 5'-GTCCCAGCACTGGTCGTTTA- 3'. GAPDH was used as internal control. The primers of GAPDH (mRNA: NM_001256799.2) were 5'-GAGAAGGCTGGGGCTCATTT-3'; 5'-AGTGATGGCATGGACTGTGG-3'. The PCR product length of SMUG1 and GAPDH were 190bp and 231 bp, respectively. All reactions were performed with a ViiA 7 Dx System (ABI). The cutoff point (Ct) was defined as the value when the fluorescent signal increased above the background threshold. The ΔCt for gene-specific mRNA expression was calculated relative to the Ct of GAPDH. Relative mRNA expression was calculated with the formula: 2-ΔCt.

SMUG1 protein expression by Western blotting

Eighty seven freshly CSCCs tissues were used to detect SMUG1 protein expression. Briefly, the tissue samples were minced on ice, and then the tissue was homogenized in the RIPA protein lysis buffer. The homogenized mixtures were rotated in the tubes at 4℃ for 1 h, and after centrifugation at 12,000 rpm at 4℃, the supernatant was collected and the protein concentrations were quantified.

10μl protein lysates were loaded into an 8% PAGE gel. Subsequently, electrophoretic separated proteins were transferred onto a 0.45µm PVDF membrane. After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000). After washing with TBS, the membranes were imaged with ECL using an Image Quant LAS 4000 mini (GE Healthcare).

Statistical Analysis

For the association between the genotypes and risk of cervical carcinoma, the odds ratio (OR), 95% confidence intervals (CIs) and P-values were obtained by binary logistic regression analysis. The control was set as the reference group for analysis. Stratified analysis of life style habits and genotype frequencies were evaluated with Kruskal-Wallis H test. The differences of quantitative mRNA and protein expression were calculated by ANOVA with a post hoc analysis (Fisher least significant difference). All reported values are two-tailed. The level of statistically significant difference was set at P≤0.05. All statistical analysis was done with SPSS software 18.0 ver for Windows.

Results

Clinical Features of Cases and Controls

40 years old/40 years old individuals were 602/598, 258/ 142 and 160/240 in the control, CIN III and carcinoma respectively. The carcinoma group had significantly more individuals >40 years old, but the CIN III group had more <40 years old individuals (P<0.001) compared to the control. There was no significant difference beside the increase of the proportion of individuals with number of parities more than 3 in the CIN III and carcinoma groups. The HR- HPV infection rate was 31.4%, 86.8% and 88.6% in the control, the CIN III and the carcinoma, respectively. HR-HPV infection in CIN III and cervical carcinoma cases were more than in controls. The Table 2 and data are quoted from our previously published work [23].

Correlation analysis of SMUG1 SNPs Genotypes with risk of CSCC

Table 3 represents the genotypic and allelic frequencies of SMUG1 rs3087404, rs2029166, rs2029167 and rs7296239. Genotype distributions were in Hardy-Weinberg equilibrium. The CC, CT, and TT frequency of SMUG1 rs2029166 was 42.1%, 46.1% and 11.6% in the controls, 40.8%, 45.3% and 14.0% in the CIN III, 43.5%, 39.8% and 16.8% in CSCCs. The TT, TC, and CC frequency of SMUG1 rs7296239 was 33.0%, 51.9% and 15.1% in the controls, 36.0%, 51.8% and 12.3% in the CIN III, 34.8%, 49.3% and 16.0% in CSCCs, respectively. These results indicated that the SMUG1 rs2029166 and rs7296239 polymorphism were not associated with the risk for CIN III or CSCCs.

The AA, AG, and GG frequency of SMUG1 rs3087404 was 34.5%, 54.9% and 10.6% in the controls, 35.3%, 45.5% and 19.3% in the CIN III, 28.3%, 36.8% and 35.0% in CSCCs, respectively. These results revealed that women with the homozygous GG of rs3087404 had a significantly increased risk of CIN III [OR=1.78(1.27-2.51), P= 0.001] and CSCC [OR=4.04 (2.94-5.55), P=0.000]. We observed “A” allele is the major form at rs3087404 in controls (62.0%, 1487/ 2400), but “G” allele is the major form in CSCCs (53.4%, 427/800). The increased risk of “G” allele in CIN III and CSCCs were 1.78(1.00-1.39) and 1.86 (1.59-2.19) respectively. Data also indicated that individuals with “G” allele or “G” carrier (AG +GG) at rs3087404 were at higher risk for CSCCs [OR=1.34 (1.04-1.71), P= 0.022].

The AA, AG, and GG frequency of SMUG1 rs2029167 was 37.9%, 44.3% and 17.8% in the controls, 30.3%, 33.5% and 36.3% in the CIN III, 24.3%, 29.8% and 46.0% in CSCCs, respectively. These results revealed that women with the homozygous GG of rs2029167 had an increased risk of CIN III [OR=2.56 (1.91-3.43), P= 0.000] and CSCCs [OR=4.05(3.02-5.44), P=0.000]. “A” allele is the major form at rs2029167 in controls (60.1%, 1442/2400), but “G” allele is the major form in CIN III (53.0%, 424/800) and in CSCCs (60.9%, 487/800). “G” allele at rs2029167 was significantly higher in CIN III and CSCCs compared with normal controls. The increased risk of “G” allele in CIN III and CSCCs were OR=1.70(1.45-1.99) and OR=2.34(1.99-2.76) respectively. “G” allele or “G” carrier (AG +GG) at rs2029167 were at higher risk for CINIII [OR=1.41(1.10-1.80), P= 0.006] and CSCCs [OR=1.91 (1.48-2.47), P= 0.000].

As show in Table 4, in the HR-HPV positive group, though the homozygous GG of rs3087404 have not increased the risk of CIN III [OR=1.43(0.80-2.53, P= 0.226], it significantly increased the risk of CSCCs [OR=3.91(2.15-7.15), P=0.000], the increased risk of “G” allele in CSCCs were OR=1.94(1.45-2.60). The homozygous GG of rs2029167 had an increased risk of CIN III [OR=2.40 (1.48-3.89), P= 0.000] and CSCCs [OR=3.88(2.26-6.88), P=0.000]. Meanwhile, the increased risk of “G” allele in CIN III and CSCCs were OR=1.66(1.29-2.15) and OR=2.35(1.75-3.15).

 Table 2 

Frequency distribution of select features by case control status

VariableControlCIN IIIχ2*PCarcinomaχ2*P
N=1200, N(%)N=400, N(%)N=400, N(%)
Age≤40602 (50.2)258(64.5)24.793<0.001160(40.0)12.431<0.001
>40598(49.8)142(35.5)240(60.0)
Age at the first intercourse≤20 years359(29.9)130(32.5)0.9430.331125(31.3)0.2530.615
>20 years841(70.1)270(67.5)275(68.8)
Number of sexual partners≤ 1963(80.3)316(79.0)0.2920.589309(77.3)1.6570.198
>1237(19.8)84(21.0)91(22.8)
Age at the first birth≤22years235(19.6)91(22.8)1.8540.17389(22.3)1.3210.25
>22 years965(80.4)309(77.3)311(77.8)
Number of parities**≤ 3548(45.7)158(39.5)4.6270.031131(32.8)20.49<0.001
>3652(54.3)242(60.5)269(67.3)
Smoking statussmoker4(0.3)2(0.5)0.2230.6372(0.5)0.2230.637
nonsmoker1196(99.7)398(99.5)398(99.5)
HR-HPV infectionPositive191(31.4)310(86.8)277.107<0.001178(88.6)199.315<0.001
Negative418(68.6)47(13.2)23(11.4)
total609357201

Bold values show statistical data with significant difference.* Two-sided χ2 test. ** Parities including full-term pregnancy and abortion at or after 28 weeks

 Table 3 

Association between SMUG1 polymorphisms and the risk of CIN III and cervical carcinoma

SMUG1 GenotypesAll patients and controls
Control
N=1200
CIN III
N=400
adjusted OR* (95% CI)PCarcinoma
N=400
adjusted OR* (95% CI)P
N%N%N%
rs3087404
AA41434.514135.31.00(ref)11328.31.00(ref)
AG65954.918245.50.81(0.63-1.04)0.10314736.80.82(0.62-1.08)0.149
GG12710.67719.31.78(1.27-2.51)0.001 14035.04.04(2.94-5.55)0.000
AG+GG78665.525964.80.97(0.76-1.23)0.78528771.81.34(1.04-1.71)0.022
Allelic frequency
Allele A148762.046458.01.00(ref)37346.61.00(ref)
Allele G91338.033642.01.78(1.00-1.39)0.047 42753.41.86(1.59-2.19)0.000
rs2029166
CC50442.016340.81.00(ref)17443.51.00(ref)
CT55746.418145.31.01(0.79-1.28)0.96915939.80.83(0.65-1.06)0.131
TT13911.65614.01.25(0.87-1.78)0.2286716.81.40(1.00-1.96)0.053
CT+TT69658.023759.31.05(0.84-1.33)0.66122656.50.94(0.75-1.18)0.599
Allelic frequency
Allele C156565.250763.41.00(ref)50763.41.00(ref)
Allele T83534.829336.61.08(0.92-1.28)0.34729336.61.08(0.92-1.28)0.347
rs2029167
AA45537.912130.31.00(ref)9724.31.00(ref)
AG53244.313433.50.95(0.72-1.25)0.70011929.81.05(0.78-1.41)0.750
GG21317.814536.32.56(1.91-3.43)0.000 18446.04.05(3.02-5.44)0.000
AG+GG74562.127969.81.41(1.10-1.80)0.006 30375.81.91(1.48-2.47)0.000
Allelic frequency
Allele A144260.137647.01.00(ref)31339.11.00(ref)
Allele G95839.942453.01.70(1.45-1.99)0.000 48760.92.34(1.99-2.76)0.000
rs7296239
TT39633.014436.01.00(ref)13934.81.00(ref)
TC62351.920751.80.91(0.71-1.17)0.47419749.30.90(0.70-1.16)0.415
CC18115.14912.30.74(0.52-1.08)0.1176416.01.00(0.71-1.42)0.967
TC+CC80467.025664.00.88(0.69-1.11)0.27226165.30.93(0.73-1.17)0.521
Allelic frequency
Allele T141559.049561.91.00(ref)47559.41.00(ref)
Allele C98541.030538.10.89(0.75-1.04)0.14532540.60.98(0.84-1.16)0.836

Bold values show statistical data with significant difference. *All P-values are adjusted for age, number of sexual partners, age at first intercourse, parities (including full-term pregnancy and abortion at or after 28 weeks) and age at first full-term pregnancy.

 Table 4 

Association between SMUG1 rs3087404 and rs2029167 polymorphisms with the risk of HR-HPV positive cervical carcinoma and CIN III

SMUG1 GenotypesHPV-positive patients and controls
Control
N=191
CIN III
N=310
adjusted OR*(95% CI)Pcarcinoma
N=178
adjusted OR*(95% CI)P
N%N%n%
rs3087404
AA6534.011135.81.00(ref)4927.51.00(ref)
AG10353.914346.10.81(0.55-1.21)0.3076134.30.79(0.48-1.28)0.332
GG2312.05618.11.43(0.80-2.53)0.2266838.23.91(2.15-7.15)0.000
AG+GG12666.019964.20.93(0.63-1.35)0.68612972.51.36(0.87-2.12)0.177
Allelic frequency
Allele A23361.036558.91.00(ref)15944.71.00(ref)
Allele G14939.025541.11.09(0.84-1.42)0.50619755.31.94(1.45-2.60)0.000
rs2029167
AA7137.29229.71.00(ref)4223.61.00(ref)
AG8343.510333.20.96(0.63-1.46)0.8415128.71.04(0.62-1.74)0.885
GG3719.411537.12.40(1.48-3.89)0.000 8547.83.88(2.26-6.88)0.000
AG+GG12062.821870.31.40(0.96-2.05)0.08313676.41.92(1.22-3.02)0.005
Allelic frequency
Allele A22558.928746.31.00(ref)13537.91.00(ref)
Allele G15741.133353.71.66(1.29-2.15)0.000 22162.12.35(1.75-3.15)0.000

Bold values show statistical data with significant difference. *All P-values are adjusted for age, number of sexual partners, age at first intercourse, parities (including full-term pregnancy and abortion at or after 28 weeks) and age at first full-term pregnancy.

Association between SMUG1 rs3087404, rs2029167 polymorphisms and the Sexual, Reproductive History in CSCCs and CIN III

As show in Table 5, the participants were divided into two groups according to Age, age of first sexual intercourse, number of sexual partners, age at first parity, number of parities and HR-HPV infection, then stratified analysis was done with the SMUG1 rs3087404 (A/G) and rs2029167 (A/G) genotype. Stratified analysis of age, number of parities, and age at first parity showed no correlation with rs3087404 (A/G) polymorphisms. However, we find a particularly high level of enrichment between groups with stratified analysis of the number of sexual partners in the CIN III (χ2=15.610, P=0.000) and CSCCs (χ2=13.468, P=0.000), and the age of first sexual intercourse in the CIN III (χ2=18.453, P=0.000) and CSCCs (χ2=15.528, P=0.000). We also did not find a high level of enrichment between HR-HPV positive and negative group of CIN III (χ2=0.176, P=0.675) and CSCCs (χ2=0.017, P=0.895).

Data display of rs2029167 (A/G) as show in Table 6, we did not find a particularly high level of enrichment between groups, except for the number of sexual partners in the CIN III (χ2=10.214, P=0.001) and CSCCs (χ2=12.366, P=0.000), there was a particularly high level of enrichment.

Association between SMUG1 rs3087404, rs2029167 polymorphisms and the Clinical pathological characteristics in CSCCs

The correlation of SMUG1 rs3087404 and rs2029167 polymorphisms with CSCCs clinicopathological characteristics is shown in Table 7.The CSCCs were divided into two groups according to age, tumor family history, FIGO stage, tumor size, differentiation grade, lymph node metastasis, vascular involvement, stromal invasion, vaginal wall extension, parametrial extension, and endometrial extension, then stratified analysis was done with the SMUG1 rs3087404 (A/G) and rs2029167 (A/G) genotype.

Stratified analysis of age, FIGO stage, tumor size, lymph node metastasis, vascular involvement, stromal invasion, vaginal wall extension, parametrail extension, and endometrial extension showed no correlation with rs3087404 (A/G) or rs2029167 (A/G) polymorphism. However, we found a particularly high level of enrichment of rs3087404 (χ2=9.265, P= 0.002)) and rs2029167 (χ2=8.112, P=0.004) when stratified by differentiation grade. This means that GG homozygotes of rs3087404 and rs2029167 are significantly associated with the degree of malignancy of tumor differentiation. In addition, interestingly, we found that GG homozygote of rs3087404 is also associated with a family history of the tumor (χ2=8.792, P=0.003).

mRNA and protein expression from patients with different genotypes of SMUG1 rs3087404 (A/G) or rs2029167 (A/G)

As shown in Figure 1, among the 87 CSCCs patients, the genotypes of AA, AG, and GG at rs3087404 were 25(28.7%), 32(36.8%) and 30(34.5%) cases, while the genotypes of AA, AG, and GG at rs2029167 were 22(25.3%), 26(30.0%) and 39(44.8%) cases, respectively. There was no significant difference in the expression of SMUG1 mRNA with different genotypes at rs3087404 (F=1.022, P=0.364) or at rs2029167 (F=2.067, P=0.133).

As shown in Figure 2, Western Blot experiments confirmed that the polymorphism of rs3087404 did not affect the expression of SMUG1 protein (F=0.254, P=0.781). Similarly, the polymorphisms of rs2029167 is also independent of the expression level of SMUG1 protein (F=1.346, P=0.308).

 Figure 1 

SMUG1 mRNA expression in CSCCs with different genotypes of rs3087404 and rs2029167 (qPCR).

J Cancer Image (Click on the image to enlarge.)
 Figure 2 

SMUG1 protein expression in CSCCs with different genotypes of rs3087404 and rs2029167 (Western Blot) (A) AA: rs3087404 genotype is AA; AG: rs3087404 genotype is AG; GG: rs3087404 genotype is GG; (B) AA: rs2029167 genotype is AA; AG: rs2029167 genotype is AG; GG: rs2029167 genotype is GG

J Cancer Image (Click on the image to enlarge.)
 Table 5 

Association between SMUG1 rs3087404 polymorphisms and the risk for CIN III and cervical carcinoma stratified by the sexual, reproductive history

High risk exposureControlsχ2 PCIN IIIχ2PCarcinoma χ2P
AAAGGGAAAGGGAAAGGG
N%N%N%N%N%N%N%N%N%
Age
≤ 4021235.232353.76711.10.0368.4909336.012046.54517.40.8370.3604427.56138.15534.40.0000.991
>4020233.833656.26010.04833.86243.73222.56928.88635.88535.4
Number of sexual partners
≤ 132133.353855.910410.82.5900.10812038.015147.84514.215.610 0.000 9330.112640.89029.113.468 0.000
>19339.212151.1239.72125.03136.93238.12022.02123.15054.9
Age at the first intercourse
≤2012234.018752.15013.91.3640.2433224.65743.84131.518.453 0.000 2620.83628.86350.415.528 0.000
>2029234.747256.1779.210940.412546.33613.38731.611140.47728.0
Number of parities
≤ 318433.629954.66511.91.0550.3045132.37446.83320.91.0940.2963426.05239.74534.40.0690.792
>323035.336055.2629.59037.210844.64418.27929.49535.39535.3
Age at the first parity
≤228435.713155.7208.50.6750.4113134.14347.31718.70.0140.9052224.73640.43134.80.1830.669
>2233034.252854.710711.111035.613945.06019.49129.311135.710935.0
HR-HPV infection status
Positive6534.010353.92312.00.5950.44011135.814346.15618.10.1760.6754927.56134.36838.20.0170.895
Negative15136.122754.3409.61634.02144.71021.3626.1939.1834.8

Bold values show statistical data with significant difference. Stratified analysis were applied by the Kruskal-Wallis H. A p value less than 0.05 was considered significant.

 Table 6 

Association between SMUG1 rs2029167 polymorphisms and the risk for CIN and cervical carcinoma stratified by the sexual, reproductive history

High risk exposureControlsχ2 PCIN IIIχ2PCarcinoma χ2P
AAAGGGAAAGGGAAAGGG
N%N%N%N%N%N%N%N%N%
Age
≤ 4023438.927846.29015.02.7020.1007529.18834.19536.80.3250.5693823.84427.57848.80.5130.474
>4022137.025442.512320.64632.44632.45035.25924.67531.310644.2
Number of sexual partners
≤ 136237.643144.817017.70.0740.78610332.611235.410132.010.214 0.001 8326.99932.012741.112.366 0.000
>19339.210142.64318.11821.42226.24452.41415.42022.05762.6
Age at the first intercourse
≤2013236.816445.76317.50.1210.7283829.24232.35038.50.3120.5762923.23628.86048.00.2740.601
>2032338.436843.815017.88330.79234.19535.26824.78330.212445.1
Number of parities
≤ 320236.923843.410819.71.5390.2154629.15132.36138.60.4940.4823123.73627.56448.90.4160.519
>325338.829445.110516.17531.08334.38434.76624.58330.912044.6
Age at the first parity
≤228837.410143.04619.60.2370.6262527.52931.93740.70.9330.3341921.32325.84752.81.7970.180
>2236738.043144.716717.39631.110534.010835.07825.19630.913744.1
HR-HPV infection status
Positive7137.28343.53719.40.0280.8679229.710333.211537.10.3300.5664223.65128.78547.80.1350.713
Negative14935.619145.77818.71634.01531.91634.0521.7626.11252.2

Bold values show statistical data with significant difference. Stratified analysis were applied by the Kruskal-Wallis H. A p value less than 0.05 was considered significant.

Haplotype Analysis between the Linkage Disequilibrium of the SMUG1 rs3087404 and rs2029167 Variants Genotypes and the Risk of CIN III and CSCCs

Since that the frequencies of both rs3087404 (A/G) and rs2029167 (A/G) genotypes change the risk of CIN III or CSCCs significantly, we further analyzed the linkage disequilibrium between rs3087404 (A/G) and rs2029167 (A/G). The frequencies of the nine haplotypes were shown in Table 8. GG (rs3087404)-GG (rs2029167) was not detected in normal control and CSCCs, except for 1 case detected in CIN III. Compared to AA (rs3087404)-AA (rs2029167), the genotype with AA-GG [OR=3.14 (1.95-5.05), P=0.000], AG-GG [OR=2.45(1.58-3.89), P= 0.000], GG-AA [OR=2.24(1.28-3.90), P=0.005], GG-AG [OR=2.58(1.54-4.32), P=0.027] significantly increased the risk of CIN III. More notably, this risk is much greater in CSCC: AA-GG [OR=7.13(4.03-12.61), P= 0.000], AG-GG [OR=7.22(4.21-12.38), P=0.000], GG- AA [OR=8.60(4.73-15.63), P=0.000], GG-AG [OR=9.64 (5.43-17.13, P=0.000]. This means that whether the rs3087404 or rs2029167 is GG homozygote, the linkage mode is at high risk. We also found that women with the AG-AG genotype had a decreased risk for CSCCs [OR=0.49(0.25-0.96), P=0.038].

Additionally, most GG (rs3087404) genotypes were linkage GG-AG (44/77, 80/140) in the CIN III and CSCC, while most GG (rs2029167) genotypes were linkage genotype AG-GG (79/145, 112/184) in the CIN III and CSCCs, respectively. These indicated that the majority of GG genotype distributions are caused by the linkage disequilibrium with the corresponding alleles.

 Table 7 

Association between SMUG1 rs3087404 and rs2029167 polymorphisms and the risk for cervical carcinoma stratified by clinical pathological characteristics

Clinical pathological characteristicsSMUG1 rs3087404χ2PSMUG1 rs2029167χ2P
AAAGGGAAAGGG
N%N%N%N%N%N%
Age
≤ 404226.36641.35232.50.0220.8823622.55232.57245.00.0030.955
>407129.68133.88836.76125.46727.911246.7
Tumor family history
Negative10829.413937.912032.78.792 0.003 8723.711330.816745.50.010.919
positive515.2824.22060.61030.3618.21751.5
FIGO stage
I9628.412336.411935.20.0020.9668424.910330.515144.71.3370.248
II1727.42438.72133.91321.01625.83353.2
Tumor size
<4cm9628.811735.112036.00.0910.7638124.39528.515747.10.4740.491
≥4cm1725.43044.82029.91623.92435.82740.3
Differentiation grade
Grade I-II10430.113137.911132.19.265 0.002 8925.710831.214943.18.1120.004
Grade III916.71629.62953.7814.81120.43564.8
Lymph node metastasis
Negative10128.113337.012534.80.0030.9538523.710729.816746.50.5990.439
positive1229.31434.11536.61229.31229.31741.5
Vascular involvement
Negative9628.112737.111934.80.0010.9798224.010430.415645.60.0170.896
positive1729.32034.52136.21525.91525.92848.3
Stromal invasion
<2/38429.810637.69232.62.3630.1247426.28429.812444.02.2630.132
≥2/32924.64134.74840.72319.53529.76050.8
Vaginal wall extension
Negative9229.012138.210432.82.2240.1367624.09229.014947.00.4380.508
positive2125.32631.33643.42125.32732.53542.2
Parametrail extension
Negative10428.813537.412233.81.8880.1698924.710729.616545.70.2490.618
positive923.11230.81846.2820.51230.81948.7
Endometrial extension
Negative10628.713937.712433.62.7670.0969124.711130.116745.31.0170.313
positive722.6825.81651.6619.4825.81754.8

Bold values show statistical data with significant difference. Stratified analysis were applied by the Kruskal-Wallis H. A P value less than 0.05 was considered significant.

 Table 8 

Genotypes and the risk of all CIN III and cervical carcinoma subjects

SMUG1 GenotypesaAll patients and controls
ControlCIN IIIadjusted ORb(95% CI)PCarcinomaadjusted ORb(95% CI)P
1200400400
N%N%N%
AA-AA15813.2399.81.00(ref)194.81.00(ref)
AA-AG17214.3379.30.87(0.53-1.44)0.589225.51.06(0.56-2.04)0.853
AA-GG847.06516.33.14(1.95-5.05)0.000 7218.07.13(4.03-12.61)0.000
AG-AA23919.95012.50.85(0.53-1.35)0.485184.50.63(0.32-1.23)0.174
AG-AG29124.35313.30.74(0.47-1.17)0.192174.30.49(0.25-0.96)0.038
AG-GG12910.87919.82.45(1.58-3.89)0.000 11228.07.22(4.21-12.38)0.000
GG-AA584.8328.02.24(1.28-3.90)0.005 6015.08.60(4.73-15.63)0.000
GG-AG695.84411.02.58(1.54-4.32)0.027 8020.09.64(5.43-17.13)0.000
GG-GG00.010.3__00.0__

Bold values show statistical data with significant difference. agenotypes are composed of two polymorphic sites: rs3087404(A/G), rs2029167(A/G). bAll P-values are adjusted for age, number of sexual partners, age at first intercourse, parities (including full-term pregnancy and abortion at or after 28 weeks) and age at first full-term pregnancy.

Discussion

Uracil misincorporation into DNA arises spontaneously at low level as a result of cytosine deamination or misincorporation of dUMP during DNA replication [24, 25]. Under normal conditions, such lesions are rapidly recovered by the BER pathway initiated by uracil-DNA glycosylases (UDG) [26, 27]. In most organisms, including humans, uracil is generally an undesirable ingredient in the genome. Thus strategies are in place to remove uracil once occurring of the DNA damage. So, sophisticated mechanisms are essential for the removal of uracil from DNA and prevention of its misincorporation, and maintain genomic integrity and stability. The failure of removing misincorporated uracil from DNA will result in base abnormity during DNA replication, even lead to dsDNA breaks and chromosomal aberrations, these two events are the key genetic factors of tumorigenesis [25, 28-30].

BER is a highly conserved DNA repair system from bacteria to humans [31-33]. A great variety of DNA-damaging agents can cause genome instability, which would be a tremendous matter for cells if the damaged DNA is not recovered [11]. The most important role of BER is to remove DNA damage caused by various carcinogens, such as reactive oxygen species (ROS), ionizing radiation and so on [34]. In humans, four UDGs have been identified, encoded by the UNG, SMUG1, MBD4 and TDG genes [33,35,36]. Most of these critical BER genes are highly polymorphic [37]. Genetic variations of these genes are likely to alter BER enzyme functional activity, and influence cancer risk [36].

The human single-strand-selective monofunctional uracil-DNA glycosylase 1(SMUG1; also named: FDG, UNG3 and HMUDG) is located in the Chromosome 12q13.11-13.3 [26, 38]. This gene encodes for a uracil DNA glycosylase (UDG) of the BER pathway that removes uracil, from single stranded (ssDNA) as well as double stranded DNA (dsDNA) [39]. As SMUG1 removes uracil and 5-hmeU from ssDNA and dsDNA, this enzyme may take participate in the repair of deamination and oxidation damage. The SMUG1 is the major enzyme involved in the removal of 5-hmeU from damaged DNA [40].

Several researchers reported the SNPs of SMUG1were correlationship with bladder cancer, breast cancer and CRC susceptibility. In a matched study of 801 bladder cancer cases, Xie et al. found 13 SNPs in10 BER pathway genes significantly increased the risk of bladder cancer. The most significant variant was SMUG1 rs2029167 (A/G). The homozygous GG genotype increased a 1.42-fold risk of bladder cancer [19].In another 1,077 case-controls matched study of incident breast cancer, Marian et al suggested that there was increased risk of breast cancer among postmenopausal women who were heterozygous of two of SMUG1 SNPs which is thought to be the most active glycosylase in vivo, raises the possibility that subtle 'heterosis' effects on cancer risk might be produced by these SNPs [20]. In a study of CRC, Broderick et al reported that genetic variations in TDG, UNG and SMUG1 may play a role in the susceptibility of CRC [21]. These reports remind us to make a hypothesis which there is an association between the genetic variants of SMUG1 gene with cancer risk. We carried out the correlation study of cervical cancer and SMUG1 SNPs.

The initiation and development of cervical carcinoma involves reversible transformation in the cervical squamous cells resulting in various cellular abnormalities and ultimately to cervical tumorigenesis. The development of cervical carcinoma usually requires multiple stages, eventually developing from precursor lesion cervical intraepithelial neoplasia (CIN) to cervical malignant carcinoma [21]. In our results, the two of SMUG1 rs2029166 and rs7296239 polymorphisms were not associated with the risk for CIN III or CSCC. Interestingly, the homozygous GG of rs3087404 and rs2029167 had a significantly increased risk of CIN III and CSCC. We also observed the increased risk of G allele of these two SNP in CIN III and CSCC. The individuals with G allele or G carrier (AG +GG) at rs3087404 and rs2029167 were at higher risk for CSCC. These findings indicated that the SMUG1 rs2029166 and rs7296239 polymorphisms (G allele) maybe play a role in initiation and progression of precancerous lesions (CIN) and cervical carcinoma. So far, there is no study about the correlation between cervical carcinoma and the SMUG1 rs2029166 and rs7296239 polymorphisms (G allele). The present study is the first time to discover the association between the SMUG1 rs2029166 and rs7296239 polymorphisms (G allele) and cervical carcinoma or CIN III. In general, SNP loci that affect the structure and function of genes are located in the 5' UTR promoter, coding region, or 3' UTR region. Although our two variants both are located in the intron which cannot change the amino acid, it is possible that there is linkage disequilibrium with other functional genetic variants and serves as a genetic marker of susceptibility [41]. Another possibility is that the SMUG1 rs2029166 and rs7296239 genetic variants maybe influence primary mRNA splicing and regulation, and affects SMUG1 protein expression or produce alternative spliceosome. To validate the SMUG1 expression change, we detected the mRNA and protein expression in fresh tumor tissues in the different genotype groups of SMUG1 rs2029166 and rs7296239, but we discovered that there was no association between the genotype of SMUG1 rs2029166 and rs7296239 with SMUG1 gene mRNA or protein expression. These indicate that the tumor susceptibility induced by the polymorphism of this locus was not achieved by altering gene expression. Bonnet et al. speculated that the introns take participate in maintaining genetic stability at certain locations, particularly in highly expressed genes [42], and repair genes are often high expression genes.

During the linkage disequilibrium analysis between rs3087404 (A/G) and rs2029167 (A/G), we found that whether the rs3087404 or rs2029167 is GG homozygote, the linkage mode is at high risk. Additionally, most GG (rs3087404) genotypes were linkage GG-AG (44/77, 80/140) in the CIN III and CSCCs, while most GG (rs2029167) genotypes were linkage genotype AG-GG (79/145, 112/184) in the CIN III and CSCCs, respectively. These indicated that the majority of GG genotype distributions are caused by the linkage disequilibrium with the corresponding alleles. These linkage modes can be used as genetic biomarker of early prediction of cervical carcinoma, as an indicator of primary prevention.

Stratified analysis of the number of sexual partners and the age of first sexual intercourse found that the rs3087404 (A/G) had a particularly high level of enrichment in the CIN III and CSCCs. About the rs2029167 (A/G), we only found a particularly high level of enrichment grouping by the number of sexual partners in the CIN III. This suggests that there may be a certain correlation between SMUG1 rs3087404 (A/G) and rs2029167 (A/G) variants with the female sexual behavior.

Among all of clinical parameters, we found that the genetic polymorphisms of rs3087404 (A/G) and rs2029167 (A/G) are significantly associated with the degree of malignancy of tumor differentiation; homozygous GG genotype increases the risk of malignant cell differentiation grade of tumors. In addition, interestingly, we found that GG homozygote of rs3087404 is also associated with a family history of the tumor. These indicate that there maybe a correlation between the SNPs of SMUG1 rs3087404 (A/G) and rs2029167 (A/G) with tumor cell differentiation and family heredity.

In HR-HPV positive group, we found that the homozygous GG of rs3087404 and rs2029167 both significantly increased the risk of CSCCs, only “G” allele or “G” carrier (AG +GG) at rs2029167 were at higher risk for CSCCs. But, in stratified analysis, we did not find a high level of enrichment between HR-HPV positive and negative groups of CIN III and CSCCs. These indicated that the rs3087404 and rs2029167 involved in the cervical tumorigenesis, but they maybe not affect the HR-HPVs infection at early onset of disease. In the process of affecting cervical tumorigenesis, rs2029167 variant may be more effective than those of rs3087404 variant.

These findings suggested that there was association between the two of SMUG1 rs3087404 (A/G) and rs2029167(A/G) genetic variant with the susceptibility of CIN III and CSCCs, but not HR-HPVs infection. Whether the rs3087404 or rs2029167 is GG homozygote, there was linkage disequilibrium between these two of polymorphism leading to increase the risk of CIN III and CSCC. These linkage modes can be used as genetic biomarker of early prediction of cervical carcinoma, as an indicator of primary prevention.

Abbreviations

CC: cervical cancer; HR-HPV: high-risk HPV; BER: base excision repair; SNP: single nucleotide polymorphisms; SMUG1: Single-strand selective mono-functional uracil-DNA glycosylase; CRC: colorectal cancer; CSCC: cervical squamous cell carcinoma; CIN: cervical intraepithelial neoplasia; MA-PCR: Modified polymerase chain reaction-mismatch amplification; HC II: Hybrid Capture II; OR: odds ratio; CIs: 95% confidence intervals; UDG: uracil-DNA glycosylases; ROS: reactive oxygen species; ssDNA: single stranded DNA; dsDNA: double stranded DNA.

Acknowledgements

The authors are grateful to Drs. Caiyun Zhou and Minhua Yu for their assistance in recruiting the subjects.

Grants

This work was supported by grants from the National Nature Science foundation of China (No. 30973380) and Zhejiang Provincial Natural Science Foundation of China (No. Y2110200).

Author Contributions

Conceived and designed the experiments: FY HC. Performed the experiments: FY HW QC JL XC. Analyzed the data: FY HC. Contributed reagents/ materials/analysis tools: FY HW QC. Wrote the paper: FY HC.

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding address Corresponding author: Huaizeng Chen, MD. (chenhzedu.cn). Women's Hospital, School of Medicine, Zhejiang University, Xueshi Rd #1, Hangzhou, 310006, Zhejiang Province, P.R. China. Phone: 86-571-87061501, Fax: 86-571-87061878


Received 2018-5-6
Accepted 2018-10-23
Published 2019-1-1