Specimens study and collection

Abstract

Background: MDM2 SNP309 (rs2279744) is a single nucleotide T>G polymorphism present in the first intron of the MDM2 gene and a negative regulator of p53 tumor suppressor protein. The findings suggest that MDM2 309TG polymorphism may be a risk factor for several cancers. This study examined clinical associations of thyroid tumors with SNP309 in Iranian-Azeri population.

Methods: In present study, 107 thyroid cancer patients and 156 cancer-free control were obtained from Iranian-Azeri population. Genomic DNA including of peripheral blood and tumor samples was extracted by salting out procedure. The MDM2 SNP309 genotyping was carried out by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) assay. All analyses were conducted by spss software with Chi-squared(χ2) test and the P < 0.05 was used as the criterion of significance. Results: Significant difference between genotype frequency distribution in control and cancerous group was found and our results showed that the genotypes containing G allele [TG (OR, 0.021; 95% CI, 0.018–0.024; p= 0.018) or GG (OR,0.01; 95% CI, 0.008–0.012; p= 0.007] compared with the TT genotype were associated with significant increased susceptibility to thyroid tumors. Conclusions: All Our findings imply that the MDM2 promoter SNP309 (rs2279744) is associated with the incidence of Thyroid tumors in Iranian-Azeri population. Key Words: Thyroid cancer,MDM2 SNP309 T>G, polymorphism

Introduction

The sequence of the whole human genome was completed in 2001 [1], and Approximately 6.5 million SNPs (single nucleotide polymorphisms) have been detected in human genes. Depending on where a SNP occurs, it might have different results at the phenotypic level. SNPs are located in the coding regions of genes that alter the function or structure of the encoded proteins and in non-coding regions of the genome, and have no direct known effect on the phenotype of an individual. These differences could contribute to many of the individual features that describe us as unique. Also, because they occur at a relatively high frequency in the genome (approximately one SNP for every 1000 bp), SNPs can be used as markers for these more important genetic changes. 89% of the analyzed SNPs are located in an exon and 11 % in an intron [2,3].

Thyroid cancer (TC) is the most common malignancy of the endocrine system and accounts for approximately 2.1% of all cancers diagnosed worldwide. The thyroid cancer has a 4.4% prevalence in women and a 1.3% prevalence in men. The male-to-female ratio was approximately 1: 3.5, while the crude incidence for men was 1.9/100,000 and that for women was 6.6/100,000. Thyroid cancer is the ninth most common cancer (2.1% of all cancers) in women [4]. The incidence rate of thyroid cancer in both women and men is increasing[5]. Primary thyroid tumors are classified as benign or malignant, which originate from follicular and parafollicular (or c-cells) epithelial cells. Benign tumors containing follicular adenoma and malignant tumors are contained papillary, follicular, medullary and anaplastic carcinomas. The follicular cells convert iodine into thyroxine (T4) and triiodothyronine (T3) and include papillary, follicular and anaplastic carcinomas and Follicular adenoma. The parafollicular or C-cells, which secrete calcitonin, contain medullary carcinoma [6]. Between thyroid tumors, papillary thyroid cancer represent approximately 80% of all thyroid malignancies [7]. Some molecularbiomarkersinvolved inthyroid tumorsincludep53, RET, BRAF, RET/PTC ,RAS, PAX8/PPARγ and NTRK1 [8].

The human homologue of the mouse double minute 2 (MDM2 or HDM2) gene locatedon chromosome 12q13-14 with 491 amino acids long and 12 exons consist of twotranscriptional promoters, constitutive promoter and p53-responsive intronic promoter [9, 10, 11]. MDM2 oncoprotein actsa critical regul-atory role for many tumor-related genes that are important for cell-cycle control such as the P53 [12]. The p53 gene is mutated in about 50% of all human cancers [13]. P53 is a tumor suppressor gene, which is involved in multiple pathways, including apoptosis, DNA repair, cell cycle arrest and senescence [14]. MDM2 and TP53 regulate each other through a feedback loop [15]. P53 induces MDM2 on the transcriptional level while MDM2 interacts through its N-terminal domain with an α-helix present in the transactivation domain of p53 with high affinity and inhibits its, As a result, prepares it for proteolytic degradation at the ubiquitination pathway [16]. The overall frequency of MDM2 gene amplification in human tumors is approximately 7.2% [17]. A recent study has shown that a MDM2 single nucleotide polymorphism in the first intron with a T to G change at the nucleotide 309 in the p2 promoter region of MDM2, so that, The presence of the mutant G-allele in cells containing SNP 309 GG increases the affinity of the transcriptional activator stimulatory protein 1 (Sp1), that regulates the basal levels of MDM2 mRNA and protein in these cells not in T/T wild cells. These higher levels of mdm2 in cells with the GG SNP309 alleles reduce the p53 apoptotic responses that occur in people in response to DNA damage and other environmental threats while in cells with the TT SNP309 alleles can increase p53 protein levels after a stress signal. Thus, in some individuals with a G/G genotype at SNP309, the percentage of cells undergoing apoptosis or cell cycle arrest in response to genotoxic stress is low [18, 19]. The MDM2 SNP309 polymorphism has been associated with several cancers, including gastric carcinoma [20], non-small-cell lung cancer [21], endometrial cancer [22], Colorectal Cancer [23], Hepatocellular Carcinoma [24], and bladder cancer [25]. In contrast, no increased risk was observed for breast cancer [26,27], ovarian cancer [28], prostate cancer [29]. In the present study, the association between the MDM2 SNP309 polymorphisms and thyroid tumors risk in the Iranian-Azeri was examined.

Materials and Methods

Specimens study and collection

In this study our patient group including of 107 subjects who were diagnosed with thyroid cancer (age range: 14-81 and mean age: 39.3) were eligible for this study. All patients with histologically confirmed primary thyroid cancer. Control group were selected randomly from 156 healthy subjects with no family history of cancer (age range: 19-79 and mean age: 40.9). A standardized questionnaire from the control group, including information on age, gender, family history of types cancer, smoking and alcohol consumption history was completed for everyone. Informed consent was earned from all participants. All cases and controls were ethnic Azari from northwest of Iran. The study protocol was approvedby the Ethics Committee of Tabriz University of the Medical Sciences research center (www.tbzmed.ac.ir/Research). Peripheral blood and tissue samples weretaken from patients who underwent surgery at Nour-Nejat and Emam-Reza hospitals of Tabriz-Iran, from 2008 to 2012.

DNA extraction and PCR amplification

Peripheral blood samples were kept in vials containing ethylene-diamine-tetra-acetic acid (EDTA), an anticoagulant. Genomic DNA was extracted from 5ml the whole blood mixed with anticoagulant using salting out procedure as described [30] and Then stored at – 20 until further use. The 194 bp fragment including of the T to G polymorphic site in the intronic promoter of MDM2 region was amplified using specific primers forward: 5′-CAAGTTCAGACACGTTCCGA-3′ and reverse: 5′-TCGGAACGTGTCTGAACTTG-3′. PCR was performed in a 25 µl reaction mixture containing 1μl template DNA (20-50ng), 2.5μl PCR buffer (10x), 0.5μl dNTPs (10mM), 0.75μl of each primers (10pmol), 0.85μl Mgcl2 (50mM), 18.45μl sterile distilled H2O and 0.2μl Taq DNA polymerase (5unit/μl), (Cinnagen, Iran). PCR amplification was carried out in a thermal cycler (Sensoquest, GmbH, Germany). The following cycling conditions were: an initialdenaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 59°C for 30 s and elongation at 72°C for 30 s and a final extension at 72°C for 10 min.

SSCP profiles

For sscp analysis, 4ml of the amplified pcr product added to 6ml of denaturing loading dye solution with an equivalent volume containing (95 % formamide, 10 mM NaOH, 20 mM EDTA, 0.05 % bromophenol blue and 0.05 % xylene cyanol). The solution was briefly vortexed and The total mixture were denatured by heating at 95°C for 10 min and each sample mixture was immediately snap-cooled on ice before loading onto the vertical electrophoresis set. 5µl of each pcr product sample are loaded onto a non-denaturing 10% polyacrylamide gel consisted of (5 ml acrylamide–bisacrylamide solution (40 %) (38:2), 3.5 ml Tris–Borate–EDTA buffer (TBE.5x), 13.5 ml deionized-distilled H2O, 300 µl ammonium persulfate (10 %, freshly prepared) and 30 µl tetramethylethylenediamine). Then gel was run in 0.6x TBE buffer for 15-17h under a constant voltage and temperature 100v cm/l and 4°c using a vertical electrophoretic apparatus (Akhtarian, Iran) and a power supplier (Apelex, France). One of the undenatured PCR products as negative control and a 50-bp DNA ladder (molecular size marker; Fermentas, USA) were loaded into the gel wells. After electrophoresis, the gel was silver stained to the following way: The gel was immersed in a tray containing solution 1 (4ml absolute ethanol 10% and 2ml acetic acid 5% with distilled water to a final volume of 400 mL was reached; fixing solution) and the tray was placed on top of a shaker to mix for 10 minutes (this step was performed two times). Then the solution 1 was removed, and the solution is a newly built 2 (0.1% silver nitrate) was added for 15-20 minutes. After, the solution 2 was poured out and briefly was rinsed gel with deionized water. At the end, the freshly built solution 3 (3 gr NaOH, 20 ml formaldehyde 10% in 180 mL distilled water) was added for 20-30 minutes. Solution 3 was used to wash unstained silver off the gel. Finally, bands clearly were created as dark brown regions on the gel [31,32]. Each banding pattern in the sscp gel was sequenced in order to confirm and identify sequence changes using the forward primer (Applied Biosystems, 3730xl DNA Analyzer, Bioneer, Korea). Sequencing results that were obtained were compared with the sequence of MDM2 available with the reference sequence (NC_000012.12) in the NCBI database (www.ncbi.nlm.nih.gov).

Statistical methods

At first, we assessed Hardy-Weinberg equilibrium (HWE) (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) for each study using Pearson’s goodness-of-fit chi-square in patient and control groups. Allele and genotype frequencies in patients and controls were compared by Pearson’s χ2-tests or Fisher’s exact testto determine whether there was any significant difference. Also crude odds ratios (ORs ) and 95% confidence interval (CIs) were used to assesse the association between MDM2 309T>G polymorphism and thyroid cancer risk. All statistical analyses were performed using SPSS software (v.16; SPSS Inc., USA) and p-Value < 0.05 were considered significant.

Results

Figure 1 depicts conformes of rs2279744 with distinct banding patterns in the region of interest, which was determined by sequencing (Fig. 2). The distribution of genotypes in the patient and control group were consistent with the Hardy-Weinberg equilibrium distribution (P = 0.54 and P = 0.30 ). The genotype distribution and allele frequencies of MDM2 promoter SNP309 polymorphisms between thyroid cancer patients and controls shown in table 1 and 2. As shown in Table 1, The genotype frequencies of MDM2 SNP309 (rs2279744) polymorphism among TT and GG homozygous and TG heterozygous individuals for the patient group were 29%, 18.7 and 52.3%, while in the healthy control group were 19.9%, 53.8% and 26.3%, respectively. The frequency of the wild-type allele T was in cases 44.7% (n=118) and in controls 55.3% (n=146). The frequency of the variant allele G was in cases 36.6% (n=96) and in controls 63.4% (n=166). Our results show that the MDM2 SNP309 TG/GG genotypes were associated between patients and controls compared with the SNP309TT homozygous with an increased risk of thyroid cancer. In addition, statistically significant difference did not observe in the allele frequency of MDM2 SNP309 between patient and control group. Furthermore, we also evaluated the association between the MDM2 SNP309 polymorphism and clinicopathological characteristics of thyroid cancer, including of age at diagnosis, tumor type, tumor size, gender, side involved, tumor stage and lymph node involvement but no significant difference was found.

Fig. 1 Genotypes of MDM2 SNP309 in apolyacrylamide gel electrophoresis after silver-stained. Lane 1, 2: TT homozygous, Lane 3, 4: TG heterozygous, Lane 5, 6: GGhomozygous, Lane 7: 194-bp dsDNAs (double strand DNA) is loaded as negative control, Lane L:50 bp DNA ladder.

Fig. 2 Sequencing results of rs2279744 containing PCR products. Arrows indicate the polymorphic site of genotypes. (1) TT homozygous, (2) GG heterozygous and (3) TG homozygous.

Variables TT Genotype

n(%) TG Genotype

n(%) GG Genotype

n(%) Total P value OR (95% CI)

Gender

Males 25 (30.1) 40 (48.2) 18 (21.7) 83 0.179 0.189 (0.142- 0.236)

Females 6 (25.0) 16 (66.7) 2 (8.3) 24

Age at diagnosis

<35 13 (25.0) 28 (53.8) 11 (21.2) 52 0.630 0.630 (0.572- 0.687) >36 18 (32.7) 28 (50.9) 9 (16.4) 55

Tumor type

Follicular adenoma 9 (31.0) 15 (51.7) 5 (17.2) 29 0.935 0.993 (0.982- 1.000)

Papillary carcinoma 19 (27.1) 37 (52.9) 14 (20.0) 70

Medullary carcinoma 1 (33.3) 2 (66.7) 0 (0) 3

Follicular carcinoma 2 (40.0) 2 (40.0) 1 (20.0) 5

Side involved

Left lobe 12 (38.7) 17 (54.8) 2 (6.5) 31 0.127 0.115 (0.077- 0.153)

Right lobe 9 (21.4) 25 (59.5) 8 (19.0) 42

Both lobe 2 (18.2) 5 (45.5) 4 (36.4) 11

Tumor size

<2 9 (22.5) 22 (55.0) 9 (22.5) 40 0.536 0.644 (0.587- 0.702) 2-4 9 (36.0) 13 (52.0) 3 (12.0) 25 >4 1 (20.0) 2 (40.0) 2 (40.0) 5

Tumor stage

I 16 (25.4) 37 (58.7) 10 (15.9) 63 0.812 0.796 (0.748- 0.844)

II 3 (37.5) 4 (50.0) 1 (12.5) 8

III 1 (50.0) 1 (50.0) 0 (0) 2

IV 3 (30.0) 4 (40.0) 3 (30.0) 10

Lymph node involvement

NX 4 (28.6) 9 (64.3) 1 (7.1) 14 0.868 0.919 (0.886- 0.951)

N0 11 (25.6) 24 (55.8) 8 (18.6) 43

N1 4 (23.5) 9 (52.9) 4 (23.5) 17

N1a 4 (40.0) 5 (50.0) 1 (10.0) 10

N1b 0 (0) 1 (100.0) 0 (0) 1

Table 1: Genotype and allele frequencies of MDM2 309 T/G

Pateints (%), n = 107 Control(%), n = 156 P value OR (95% CI)

General genotype

TT 31 (29) 31 (19.9) Reference

TG 56 (52.3) 84 (53.8) 0.018 0.021 (0.018-0.024)

GG 20 (18.7) 41 (26.3) 0.007 0.01(0.008-0.012)

Allele frequency

T 118 (44.7) 146 (55.3) Reference

G 96 (36.6) 166 (63.4) 0.06 0.71 (0.50-1.01)

HWE 0.549158 0.309502

Table 2: Clinicopathological characteristics of patients and controls with thyroid tumors

Discussion

MDM2 SNP309 (rs 2279744) is highly expressed in numerous types of human tumors [33]. MDM2 protein regulates p53 function in multiple ways. MDM2 directly binds with high affinity, to the N-terminal transcription domain of p53, and inhibiting its transcriptional. Furthermore, MDM2 is an E3 ubiquitin ligase that ubiquitylates, thereby promoting degradation of p53 [9, 34]. P53 is found to be mutated in 50% of human cancers and only 10% of thyroid carcinomas [35]. MDM4 possesses a high degree of homology to MDM2, especially in its N-terminal p53 binding domain [36]. MDM4 directly binds to the transactivation domain of p53 and inhibits its activity, but does not induce p53 degra-dation [14]. Under some conditions, Mdm2 can also ubiquitylate itself and Mdm4, which causes to the p53 stability and activity [37]. MDM2 also interacts with a number of other proteins, such as RYBP, RPs, ARF and PML proteins that inhibit of MDM2-mediated p53 ubiquitin/degrading and stabilization of p53 while RNF2 promotes p53 degradation [36, 38].

In some previous studies, the MDM2 SNP309 was associated with the development of cancers, whereas several studies reported no significant associations between the SNP309 and cancer risk that mentioned to some of them []. A study of meta-analysis by Gui et al. on the association between MDM2 309 T/G polymorphism and lung cancer risk among Asians in 2009 took that its results representa low proportion of MDM2 309G allele for developing lung cancer was in Asians [39]. A study by Campbell et al. indicated that the GG genotype of the MDM2 SNP309 polymorphism was not associated with either breast cancer (OR 1.04; 95% CI, 0.67–1.60) or ovarian cancer (OR 0.86; 95% CI, 0.53–1.37) and association was not found with early onset or familial breast cancer or with ovarian cancer [23]. Liu et al seven studies with a total of 4,993 subjects suggested that MDM2 rs2279744 polymorphism was significantly associated with increased risk of hepatocellular carcinoma in East Asians [40]. Another study by Sun et al. revealed that the homozygous MDM2 SNP309GG genotype simultaneously affected the risk and the onset age of breast cancer in the Taiwanese population [41]. Firoz et al. suggested that MDM2 may play an important role for the development of melanoma in women. Furthermore, showed that the women with a GG genotype were significantly higher risks of being diagnosed with melanoma at ages <50 years (With frequency of 42%) compared with women >50 years (With frequency of 14%), thereby, The MDM2 SNP309 genotype may help identify women at risk of developing melanoma at a young age [42].

In the present study, we investigated whether the common polymorphism in MDM2 SNP309 would affect risk of in samples from patients with thyroid tumors and from healthy individuals in the Iranian-Azeri population. We also invaluated the distribution of genotypes and frequency of alleles in groups of patients suffering from thyroid tumors according to different cancer grading by TNM classification [43]. Our study revealed significant association between the MDM2 SNP309 polymorphism and genotype frequency distribution in thyroid tumors so that, TG/GG genotypes were associated with a significantly increased risk for thyroid tumors (with p=0.018 and p=0.007, respectively). No significant difference in the allele frequency between cases and controls was detected. Bond and et al proved that average age of onset of cancer is low in individuals with G/G genotype and low wild-type p53 activity compared to individuals with T/T genotype [18, 19]. Similarly, we observed the influence of the G allele on the median age of onset of thyroid cancer, so that the average age of onset of cancer GG, TG and TT genotypes was 37.8, 39.75 and 41, respectively. Therefore, these results confirm the influence of the G allele on the median age of onset of thyroid cancer. The obtained data suggest that TG and GG genotypes of MDM2 SNP309 are associated with the thyroid cancers risk. Thus far, there was any study on the association between MDM2 SNP309 polymorphism and thyroid tumors risk in Iranian-Azeri population. Finally, it is assumption that the SNP309 polymorphism of the MDM2 gene may be used as a molecular biomarker to predict thyroid tumors prognosis in Iranian-Azeri population. We recommend that further studies performed on a larger group to prove this issue, as well as in other Iranian ethnic populations is also suggested.

Acknowledgments

Work of the authors laboratory is supported by radiobiology laboratory of the biology department, faculty of natural science, Tabriz University and cellular and molecular laboratory of the biology department, faculty of science, Razi University.

Conflicts of interest

The authors declare that they have no competing interestsfor this paper.

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