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.