ESR1 single nucleotide polymorphism rs1062577 (c.*3804T > A) alters the susceptibility of breast cancer risk in Iranian population

Gene 611 (2017) 9–14

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Research paper

ESR1 single nucleotide polymorphism rs1062577 (c.*3804T N A) alters the susceptibility of breast cancer risk in Iranian population Zahra Dehghan a,1, Samira Sadeghi b,1, Hossein Tabatabaeian b, Kamran Ghaedi c,d,⁎, Mansoureh Azadeh e, Mohammad Fazilati a, Fatemeh Bagheri f a

Department of Biochemistry, Payame Noor University of Isfahan, Isfahan, Iran Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran d Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran e Zist-Fanavari Novin Biotechnology Institute, Isfahan, Iran f Biochemistry Division, Department of Biology, Faculty of Science, Payame Noor University of Taft, Yazd, Iran b c

a r t i c l e

i n f o

Article history: Received 5 January 2017 Received in revised form 9 February 2017 Accepted 10 February 2017 Available online xxxx Keywords: Breast cancer ESR1 microRNA-related SNP rs1062577 Tetra-primer ARMS-PCR miR-186 miR-3636 miR-3662

a b s t r a c t Objectives: Albeit single nucleotide polymorphisms related to ESR1 gene have been studied, only a number of them have been reported to be associated with breast cancer risk. rs1062577 is one of the most recent microRNA-related ESR1 SNPs; however, no study has been conducted to investigate the significance this polymorphism in Iranian population. In this study, we aimed to investigate the frequency and also the association between rs1062577 and breast cancer. Materials and methods: rs1062577 position was genotyped by Tetra-primer ARMS-PCR in totally 182 blood specimens obtained from breast cancer patients (n = 86), and healthy blood donors (n = 96). The distribution of different genotypes was statistically analyzed in terms of the potential association between rs1062577 different alleles, breast cancer risk and clinicopathological criteria of breast cancer patients. Results: The statistical analyses confidently indicated that rs1062577 A allele is associated with the increased breast cancer risk in both univariate and multivariate regression models (Odds Ratio = 8.403 and 32.602 respectively). rs1062577 T allele was statistically associated with stage I of breast cancer patients (p-value = 0.025). In silico studies implied that rs1062577 A allele can alter the binding capacity of ESR1 mRNA and miRNAs via either breakage or formation of hydrogen bonds. Conclusion: rs1062577 A allele is significantly and dramatically associated with the elevated risk and greater stages of breast cancer. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Breast cancer, as the most widespread malignancy in females, is the first cause of cancer-related death in women with the annual 521,900 estimated death rate in the world (Torre et al., 2015). Along with other important breast cancer-related genes, including HER2, BRCA1, BRCA2, CHEK2, PTEN, TP53 and ATM (Lalloo and Evans, 2012;

Abbreviations: ATM, ataxia-telangiectasia mutated; BRCA1, BReast CAncer gene 1; BRCA2, BReast CAncer gene 2; CHECK2, checkpoint kinase 2; ER, estrogen receptor; ESR1, estrogen receptor 1; HER2, human epidermal growth factor 2; PR, progesterone receptor; PTEN, phosphatase and tensin homolog; SNP, single nucleotide polymorphism; T-ARMS-PCR, tetra-primer amplification refractory mutation system polymerase chain reaction; TP53, tumor protein p53. ⁎ Corresponding author at: Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, 81746-73441 Isfahan, Iran. E-mail address: [email protected] (K. Ghaedi). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2017.02.016 0378-1119/© 2017 Elsevier B.V. All rights reserved.

Tabatabaeian and Hojati, 2013; Hojati et al., 2014; Sadeghi et al., 2016), Estrogen Receptor 1 (ESR1) is also a very well-known proto-oncogene, associated with breast tumorigenesis (Key et al., 2002). Human ESR1 gene encodes for an estrogen receptor alpha (ERα) protein and is located on chromosome 6 (6q25.1), (Zuppan et al., 1991; Zheng et al., 2009). Deregulated ERα can function as a transcription factor regulating expression of the genes involved in growth, differentiation, invasion, and angiogenesis of malignant tumor (Gross and Yee, 2002). In addition, ERα plays a key role in pathological processes including breast cancer, osteoporosis and endometrial cancer. More than two-thirds of breast cancers show ERα overexpression, which is routinely targeted via endocrine therapy (anti-estrogen receptor) in breast cancer (Mangelsdorf et al., 1995). The ERα expression level is regulated in all transcriptional, post-transcriptional and post-translational steps (Cheng et al., 2010). MicroRNAs (miRNAs) are one of the most significant molecules regulating ESR1 expression post-transcriptionally (Pandey and Picard, 2009; Zhao et al., 2011).

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miRNAs are short non-coding RNAs of 18–23 nucleotides, negatively regulate gene expression by binding to their target mRNAs and have a dramatic impact on biological pathways such as proliferation, cell cycle control, apoptosis, migration differentiation, and metabolism (Garofalo et al., 2010). miRNAs mainly control the expression level of target genes through binding to 3′-Untranslated Region (3′UTR) of the transcripts (Garofalo et al., 2010). Consequently, these bindings cause either mRNA degradation or repression of translation process (Pillai, 2005). Aberrant expression and/or gain/loss of function of miRNAs have been reported in many studies (Palanichamy and Rao, 2014; Noormohammad et al., 2016). Among various alterations in DNA, Single Nucleotide Polymorphism (SNP) located on 3′UTR of the transcripts has drawn the attentions. These single nucleotide substitutions in DNA can lead to increase or decrease in the binding affinity of miRNAs to their target mRNAs (Chen et al., 2008; Landi et al., 2008). Recently, a number of studies on ESR1 gene SNPs have been performed including the SNPs located within its 3′UTR region, such as rs3798577, rs2747648, and rs1062577 (Li et al., 2010). The SNPs rs2747648 and rs1062577 are likely to be involved in miRNA binding affinity. Although adequate data have shown that SNP rs2747648 has an impact on breast cancer, the association between SNP rs1062577 and cancer has been reported just in a Chinese population (Chen et al., 2016). Therefore, more population-based studies are required to prove the association between rs1062577 and breast cancer. To this aim, the current study was established to investigate the frequency of SNP rs1062577 in Iranian population, and also to analyze its allele frequencies with the clinical and pathological characteristics of patients. The results showed that rs1062577 A allele was significantly associated with the enhanced risk of breast cancer in Iranian studied population.

The primer1 software was recruited to design the T-ARMS PCR primers (http://primer1.soton.ac.uk/primer1.html). To diminish any thermodynamically stable primer-dimers, duplexes, and hairpins, the primers were analyzed by Oligo 7 software (Molecular Biology Insights, Inc. DBA Oligo, Inc.). In Tetra-primer ARMS-PCR (T-ARMS PCR), two outer and two inner primers are required to specify the genotypes (Honardoost et al., 2014). The sequences of the designed primers are as follow: Outer Forward: 5′-GTTAATTATGCTCTGTTTCCAACT-3′, Outer Reverse: 5′-GCACAGCTCTAAACACACACA-3′, Inner Forward: 5′TTGAGATTCAAGAAAAATTTCTATACA-3′, and Inner Reverse: 5′CACAATTGGATGCAAAAATAA-3′. Inner forward and Inner reverse primers were respectively used to amplify rs1062577 A and T alleles. The generated product sizes for A allele, T allele, and two outer primers were 119, 178 and 251 bp respectively.

2. Material and method

2.4. Genotyping rs1062577 by T-ARMS PCR

2.1. Patients

T-ARMS PCR reactions were accomplished in a final volume of 25 μl, containing 150 ng genomic DNA, 3.5 μl 10× solution buffer, 1.5 μl dNTPs (10 mM), 1.5 μl MgCl2 (100 mM), 0.25 μl 5 U/μl Taq DNA polymerase (Bioron, Germany) and sufficient concentrations of designed primers. To optimize the multiplex-PCR circumstance, two different gradients

182 samples were included in this study, based on the random selection of the 86 breast cancer patients and 96 healthy volunteers who routinely recourse to Seyed-ol-Shohada Hospital of Isfahan for the normal

checkups. Peripheral blood samples were obtained from volunteers and kept in EDTA-tubes. The sampling procedure was performed randomly without any information about clinicopathological characteristics and also the familial background of the blood donors. This study was ethically approved according to the criteria of Iranian ministry of health and medical education. The characteristics of the studied samples and their univariate analysis are listed in Table 1. 2.2. DNA isolation To isolate the genomic DNA from the blood samples, PrimePrep Genomic DNA Isolation Kit (GeNetBio, Chungnam, South Korea) was recruited. The yielded DNA was diluted in 0.5 M TE and stored at −20 °C. 2.3. Primers

Table 1 Univariate logistic regression comparing controls and cases. Cancer (n = 86)

Controls (n = 96)

Odds ratio

p-Valuea

95% CI

Yes No Yes No

52.88(11.92) 13 73 17 69

40.31(16.82) 5 91 13 83

1.058 3.241

1.035–1.081 1.105–9.510

b0.001⁎ 0.025⁎

–

–

0.258

Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No

67 19 12 74 2 84 16 70 43 43 17 69 6 80 17 69

72 24 12 84 3 93 18 78 49 47 21 75 7 89 18 78

–

–

0.654

–

–

0.772

–

–

0.742

–

–

0.980

–

–

0.888

–

–

0.527

–

–

0.934

–

–

0.862

Variable Sociodemographic Factors Age (SD) Family history Occupation

Reproductive Factors Ever married Age at First Full-term Pregnancy N 30 Age of menarche ≤ 12 Irregular menses Age at menopause N 50 Use of Hormone Replacement Therapy Abortion Body Mass Index ≥ 30 kg/m2

a Simple Conditional Logistic Regression. Family history (No), Occupation (No), Ever married (No), Age at FFTP N 30 (No), Age of menarche ≤ 12 (No), Irregular menses (No), Age at menopause N 50 (No), Use of HRT (No), Abortion (No) and BMI ≥ 30 Kg/m2 (No) were considered as a reference of getting cancer outcome. ⁎ Statistically significant.

Z. Dehghan et al. / Gene 611 (2017) 9–14

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of the temperature (from 52 to 56 °C) and MgCl2 concentration (2 mM to 4 mM) were performed. The appropriate condition for amplification was as follow: 5 min the initial denaturing step of 95 °C, 30 cycles constituting the melting step at 94 °C for 35 s, annealing step in 56 °C for 35 s, the elongation step in 72 °C for 40 s, and the final elongation step in 72 °C for 7 min. The PCR products for different genotypes were separated and analyzed using 2.5% agarose gel electrophoresis and ethidium bromide staining. Thirty DNA samples of different genotyped DNA samples, including 13 A/A homozygotes, 17 A/T heterozygotes were sent for the Sanger sequencing to Bioneer (Cheongwon, South Korea). The provided sequencing outcomes were assessed by Chromas Lite software (Version 2.0, Technelysium Pty Ltd.). 2.5. Statistical analysis Statistical Package for Social Science (SPSS) software, version 19.0 (SPSS Inc. IBM, Chicago, USA) The different associations between the controls and breast cancer cases were analyzed for the categorical and numerical variables by univariate logistic regression. Furthermore, the breast cancer risk in the studied samples was also analyzed by the multiple logistic regression model, adjusted for all variables. The association between different genotypes of rs1062577 in breast cancer group, including A/A and A/T, and the characteristics of breast cancer was investigated using Fisher's exact test. Odds Ratio (OR) with 95%CI was reported to show the risk and p-values equal and b0.05 were considered as significant. 2.6. Data sources To have an overview about rs1062577, Minor Allele Frequency (MAF) and also flanking regions of this SNP was captured from NCBI build 37.3 databases. To analysis the interaction between the miRNA(s) and SNP-containing 3′UTR of ESR1, miRNASNP database v2.0 was enrolled to predict the potential miRNAs with the capacity to target 3′ UTR of ESR1 transcripts (Gong et al., 2012). This database was further used to show the impact of rs1062577 different alleles in modifying the affinity between ESR1 3′UTR and predicted miRNAs (either loss or gain), and also the alterations in Gibbs free energy (ΔG) of binding reaction of predicted miRNAs and 3′UTR of ESR1 mRNA. 3. Result 3.1. rs1062577 successfully genotyped by T-ARMS-PCR Based on the methodological details described in Sections 2–4, the best circumstance was used to perform multiplex PCR in one vial, using 4 different primers. It successfully displayed the A/A and A/T genotypes generated by our optimized T-ARMS PCR system, while we did not observe any T/T genotype (Fig. 1). To evaluate the plausibility, sensitivity and accuracy of T-ARMS PCR outcomes, thirty DNA specimens, including 13 A/A and 17 A/T genotypes, were sent for sequencing and all the results were quite compatible and consistent with the optimized T-ARMS PCR method. 3.2. rs1062577 A/A and A/T genotypes were detected in the studied population Genotyping of 96 normal healthy samples and 86 breast cancer patients implicated the frequency of ~ 10% for A/T genotype (18 out of 182). The distribution of A/T genotype in normal and cases was 16 and 2 respectively, reflecting the greater presence of T allele in normal healthy samples. T/T genotype was not detected in the studied population. According to the Univariate regression model, harboring T allele in rs1062577 position was significantly associated with the decreased risk of breast cancer. In another word, the breast cancer patients carried

Fig. 1. Optimized T-ARMS-PCR to genotype rs1062577. Lane 1 and 2 are respectively illustrating A/A and A/T genotypes (Product size for A allele, T allele and control band is 119, 178 and 251 bp respectively).

rs1062577 A allele were significantly displayed the higher breast cancer risk with an odds ratio of 8.403, 95%CI (1.873–37.037), (Table 2). Furthermore, harboring T allele, as the good prognostic parameter in this study, was also associated with Stage I of breast cancer (Fisher's Exact Test, p-value = 0.025). However, other clinicopathological features of the breast cancer cases showed no significant association, Table 3. To adjust the potential role of confounding variables including the risk factors listed in Table 1, the association of rs1062577 A allele-carrier genotypes with breast cancer was analyzed in a multivariate logistic regression model. This adjusted predictor model indicates that harboring the A allele in rs1062577 position is significantly associated with a drastically increased risk of breast cancer, odds ratio: 32.602 (95%CI: 5.037– 211.013), Table 4. 3.3. rs1062577 different alleles could change the affinity of interaction between ESR1 3′UTR and miRNAs According to the location of rs1062577 in 3′UTR of ESR1 gene, the potential alteration in the activity of ESR1-targeting miRNA(s) could be the noticeable mechanism of action for rs1062577. To assess this hypothesis and to investigate the potential changes in the interaction between ESR1 mRNA and its 3′UTR-targeting miRNAs, the miRNASNP database was recruited. According to the in silico analyses, rs1062577 A allele can lead to a more stable interaction between ESR1 mRNA and miR-3636 and miR-3662 via formation of new hydrogen bonds, while it may reduce the stability and affinity of the interaction between this transcript and miR-186 through the hydrogen bond breakage. These alterations in the binding affinity of miRNAs could change the regulation and activity of ESR1 gene in the cells. However, further in vitro investigations, like luciferase reporter assay, are required to validate the Table 2 Univariate logistic regression comparing T allele-harboring subjects in controls and cases. p-Valuea

Genotype Cases (n = 86)

Controls (n = 96)

Odds ratio

95% CI

A/A A/T T/T

80 (100%) 16 (0%) 0 (0%)

8.403

1.873–37.037 0.001⁎

84 (74%) 2 (26%) 0 (0%)

a Simple Conditional Logistic Regression. rs1062577 A/T genotype was considered as a reference of getting cancer outcome. ⁎ Statistically significant.

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Table 3 The association between C allele harboring patients and breast cancer characteristics. Characteristic

Status

A/A Genotype

A/T Genotype

p-Valuea

HER2

Positive Negative Positive Negative Positive Negative Positive Negative Grade I Other Grade II Other Grade III Other Stage I Other Stage II Other Stage III Other Stage IV Other Ductal Lobular

20 64 63 21 59 25 30 54 15 69 39 45 27 57 12 72 17 67 11 73 44 40 59 25

2 0 1 1 1 1 0 2 1 1 1 1 0 2 2 0 0 2 0 2 0 2 2 0

0.063

ER PR Metastasis Grade

Stage

Type

0.448 0.516 0.540 0.339 0.999 0.999 0.025⁎ 0.999 0.999 0.236 0.999

a Fisher's Exact Test. ⁎ Statistically significant.

proposed interactions. The characteristics of the proposed interactions between ESR1 3′UTR and potential miRNAs are listed in Table 5. 4. Discussion ESR1 gene, which is known and categorized as a susceptibility gene in breast cancer, have been studied yet. This gene is dramatically important due to its diverse roles in provoking the proliferation rate in mammary tissue, which in turn, increases the breast tumorigenesis (Parker et

al., 1997). Single nucleotide polymorphism located in different regions of ESR1 gene have shown the association with both risk and clinicopathological characteristics of breast cancer (Li et al., 2010; Li and Xu, 2012; Lipphardt et al., 2013). Currently, the population studies on SNPs located miRNA target sites have drawn attentions. In other words, genetic alterations within the 3′ UTR of transcribed genes have been demonstrated to modulate the affinity between miRNA and mRNAs. These single nucleotide substitutions can noticeably dysregulate the expression level of proteins, due to their functional roles in manipulating the interaction between miRNA and their target sequences. Therefore, miRNA-related SNPs can be the potential factors associated with elevated or diminished breast cancer risk (Chen et al., 2008; Landi et al., 2008; Nicoloso et al., 2010; Moradi et al., 2016). In 2008, Tchatchou, et al. reported that rs2747648 which is located within 3′UTR of ESR1 gene is significantly associated with the risk of breast cancer. This SNP might functionally change the interaction capacity between miR-453 and ESR1 transcript (Tchatchou et al., 2009). rs1062577 is another miRNA-related SNP in ESR1 gene. Our in silico analyses showed that different alleles of this position are correlated with a binding affinity of three miRNAs, miR-186, miR-3646 and miR3662. rs1062577 A allele attenuates the binding capacity of miR-186, while it renders the complementary nucleotide which is required for binding to miR-3646 and miR-3662 seed regions. Statistical analysis of our study indicated that breast cancer cases were carriers of rs1062577 A allele more often than healthy samples. In fact, A allele was significantly associated with elevated breast cancer risk, with a noticeable odds ratio of 32.602, 95%CI (5.037–211.013), in a multivariate predictor regression model. We also showed that breast cancer patients harboring T allele were entirely categorized in stage I group (Table 3), suggesting that rs1062577 T allele could be known as the good prognostic factor. Therefore, rs1062577 A allele is significantly a breast cancer risk factor in the studied population. This phenomenon can be interpreted by the attenuating effect of A allele on miR-186:ESR1 3′ UTR interaction. miR-186 has been reported to work as a tumor suppressor in a variety of human cancers including non-small cell lung

Table 4 Multivariate logistic regression comparing controls and cases. Cancer (n = 86)

Controls (n = 96)

Odds ratio

95% CI

p-Valuea

Yes No Yes No

52.88 (11.92) 13 73 17 69

40.31 (16.82) 5 91 13 83

1.070 3.241 – – –

1.044–1.098 4.515–1.167 – – –

b0.001⁎ 0.029⁎

Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No

67 19 12 74 2 84 16 70 43 43 17 69 6 80 17 69

72 24 12 84 3 93 18 78 49 47 21 75 7 89 18 78

– – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – –

A/A A/T

84 2

80 16

32.602

5.037–211.013

Variable Sociodemographic Factors Age Family history Occupation

Reproductive Factors Ever married Age at First Full-term Pregnancy N 30 Age of menarche ≤ 12 Irregular menses Age at menopause N 50 Use of Hormone Replacement Therapy Abortion Body Mass Index ≥ 30 kg/m Genetic Variation rs1062577 SNP Genotype

2

0.054

0.890 0.860 0.582 0.990 0.946 0.508 0.300 0.918

b0.001⁎

a Multiple Logistic Regression. Adjusted for age, family history, occupation, marriage, age at FFTP, age of menarche, irregular menses, age at menopause, use of HRT, abortion and BMI. ⁎ Significant predictors of breast cancer risk.

Z. Dehghan et al. / Gene 611 (2017) 9–14

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Table 5 In silico prediction of ESR1 mRNA:miRNA interactions and their characteristicsa.

miRNA

miRNA:ESR1 mRNA structure rs1062577 A allele

rs1062577 T allele

ΔG changes (A allele/T allele)

miR–186

–11.90/–13.10

miR–3646

–11.00/0.00

miR–3662

–14.90/0.00

a

The exact SNP position is underlined and the breakage of hydrogen bonds is shown by a cross.

cancer (Huang et al., 2016), bladder cancer (Yao et al., 2015) and prostate cancer (Hua et al., 2016). ESR1 might be one of the downstream targets of this tumor suppressive miRNA, and substitution of T with A allele in rs1062577 position might disturb this negative regulatory effect on ESR1 transcripts. It, in turn, up-regulates ESR1 protein and develops the ESR1-dependent proliferation rate of breast cancer cells. However, the biochemical in vitro investigations such as reporter assay, miR-186 knock-down studies in the presence and absence of A allele, and also functional studies like proliferation and apoptosis assays are intensively recommended to be performed in order to validate the postulated interaction. Furthermore, studying rs1062577 SNP in a larger sample population, and studying more parameters like BRCA1/2 status and the expression level of proposed miRNAs, in rs1062577 A and T groups, are highly recommended to improve the outcomes of this study, Briefly, our results have displayed the association between the decreased risk and predisposition of breast cancer and rs1062577 T allele. Moreover, rs1062577 T allele depicted the chance of lower breast cancer stage, within patient group. The outcome of this paper was consistent with the population-based study conducted in Han Chinese women (Chen et al., 2016), where it has been indicated that AA genotyped samples displayed increased breast cancer risk. Consequently, these two studies support the idea that rs1062577 might be proposed and nominated as a breast cancer predisposition biomarker. 5. Conclusion This study demonstrated that rs1062577 A allele is significantly and dramatically associated with the increased risk of breast cancer. This might happen through disturbing the interaction affinity between ESR1 mRNA and related miRNAs, most importantly miR-186. Our findings formed a hypothesis that rs1062577 might be proposed and considered as the potential biomarker for predisposition of breast cancer, at least in Iranian population. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment We kindly appreciate Seyed-ol-Shohada Hospital personnel for their obliging contributions.

References Chen, K., Song, F., Calin, G.A., Wei, Q., Hao, X., Zhang, W., 2008. Polymorphisms in microRNA targets: a gold mine for molecular epidemiology. Carcinogenesis 29, 1306–1311. Chen, L., Kang, H., Jin, G.-J., Chen, X., Zhang, Q.-Y., Lao, W.-T., Li, R., 2016. The association between a novel polymorphism (rs1062577) in ESR1 and breast cancer susceptibility in the Han Chinese women. Gynecol. Endocrinol. 1–4. Cheng, L., Huang, C., Ye, Q., 2010. Molecular mechanisms of regulation of estrogen receptor a expression level in breast cancer. Yi Chuan 32, 191–197. Garofalo, M., Condorelli, G., Croce, C., Condorelli, G., 2010. MicroRNAs as regulators of death receptors signaling. Cell Death Differ. 17, 200–208. Gong, J., Tong, Y., Zhang, H.M., Wang, K., Hu, T., Shan, G., Sun, J., Guo, A.Y., 2012. Genomewide identification of SNPs in microRNA genes and the SNP effects on microRNA target binding and biogenesis. Hum. Mutat. 33, 254–263. Gross, J.M., Yee, D., 2002. How does the estrogen receptor work? Breast Cancer Res. 4, 1. Hojati, Z., Hallajian, Z., Esmaeili, A., Motovali-Bashi, M., Tabatabaeian, H., 2014. Analysis of HER2 gene amplification using Differential PCR in breast cancer patients of Isfahan Province. Res. Mol. Med. 2, 12–17. Honardoost, M.A., Tabatabaeian, H., Akbari, M., Salehi, M., 2014. Investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR, based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients. Gene 549, 1–6. Hua, X., Xiao, Y., Pan, W., Li, M., Huang, X., Liao, Z., Xian, Q., Yu, L., 2016. miR-186 inhibits cell proliferation of prostate cancer by targeting GOLPH3. American Journal of. Cancer Res. 6, 1650. Huang, T., She, K., Peng, G., Wang, W., Huang, J., Li, J., Wang, Z., He, J., 2016. MicroRNA-186 suppresses cell proliferation and metastasis through targeting MAP3K2 in non-small cell lung cancer. Int. J. Oncol. 49, 1437–1444. Key, T., Appleby, P., Barnes, I., Reeves, G., 2002. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J. Natl. Cancer Inst. 94, 606–616. Lalloo, F., Evans, D., 2012. Familial breast cancer. Clin. Genet. 82, 105–114. Landi, D., Gemignani, F., Barale, R., Landi, S., 2008. A catalog of polymorphisms falling in microRNA-binding regions of cancer genes. DNA Cell Biol. 27, 35–43. Li, L.-W., Xu, L., 2012. Menopausal status modifies breast cancer risk associated with ESR1 PvuII and XbaI polymorphisms in Asian women: a HuGE review and meta-analysis. Asian Pac. J. Cancer Prev. 13, 5105–5111. Li, N., Dong, J., Hu, Z., Shen, H., Dai, M., 2010. Potentially functional polymorphisms in ESR1 and breast cancer risk: a meta-analysis. Breast Cancer Res. Treat. 121, 177–184. Lipphardt, M.F., Deryal, M., Ong, M.F., Schmidt, W., Mahlknecht, U., 2013. ESR1 single nucleotide polymorphisms predict breast cancer susceptibility in the central European Caucasian population. Int. J. Clin. Exp. Med. 6, 282–288. Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., 1995. The nuclear receptor superfamily: the second decade. Cell 83, 835–839. Moradi, B., Tabatabaeian, H., Sadeghi, S., Azadeh, M., Ghaedi, K., 2016. HER4 rs1595065 3′ UTR Variant is a Possible Risk Factor for HER2 Positivity Among Breast Cancer Patients. Thrita. Nicoloso, M.S., Sun, H., Spizzo, R., Kim, H., Wickramasinghe, P., Shimizu, M., Wojcik, S.E., Ferdin, J., Kunej, T., Xiao, L., 2010. Single-nucleotide polymorphisms inside microRNA target sites influence tumor susceptibility. Cancer Res. 70, 2789–2798. Noormohammad, M., Sadeghi, S., Tabatabaeian, H., Ghaedi, K., Talebi, A., Azadeh, M., Khatami, M., Hidari, M.M., 2016. Upregulation of miR-222 in both Helicobacter pylori-infected and noninfected gastric cancer patients. J. Genet. 95, 991–995. Palanichamy, J.K., Rao, D.S., 2014. miRNA dysregulation in cancer: towards a mechanistic understanding. Front. Genet. 5, 54.

14

Z. Dehghan et al. / Gene 611 (2017) 9–14

Pandey, D.P., Picard, D., 2009. miR-22 inhibits estrogen signaling by directly targeting the estrogen receptor α mRNA. Mol. Cell. Biol. 29, 3783–3790. Parker, M.G., Cowley, S.M., Heery, D., Henttu, P., Kalkhoven, E., Sjöberg, M., Valentine, J.E., White, R., 1997. Function of estrogen receptors in breast cancer. Breast Cancer 4, 204–208. Pillai, R.S., 2005. MicroRNA function: multiple mechanisms for a tiny RNA? RNA 11, 1753–1761. Sadeghi, S., Hojati, Z., Tabatabaeian, H., 2016. Co-overexpression of EpCAM and c-myc genes in malignant breast tumors. J. Genet. Tabatabaeian, H., Hojati, Z., 2013. Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using real time RT-PCR and immunohistochemistry. Gene 531, 39–43. Tchatchou, S., Jung, A., Hemminki, K., Sutter, C., Wappenschmidt, B., Bugert, P., Weber, B.H., Niederacher, D., Arnold, N., Varon-Mateeva, R., 2009. A variant affecting a putative miRNA target site in estrogen receptor (ESR) 1 is associated with breast cancer risk in premenopausal women. Carcinogenesis 30, 59–64.

Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., Jemal, A., 2015. Global cancer statistics, 2012. CA Cancer J. Clin. 65, 87–108. Yao, K., He, L., Gan, Y., Zeng, Q., Dai, Y., Tan, J., 2015. MiR-186 suppresses the growth and metastasis of bladder cancer by targeting NSBP1. Diagn. Pathol. 10, 146. Zhao, Y., Deng, C., Wang, J., Xiao, J., Gatalica, Z., Recker, R.R., Xiao, G.G., 2011. Let-7 family miRNAs regulate estrogen receptor alpha signaling in estrogen receptor positive breast cancer. Breast Cancer Res. Treat. 127, 69–80. Zheng, W., Long, J., Gao, Y.-T., Li, C., Zheng, Y., Xiang, Y.-B., Wen, W., Levy, S., Deming, S.L., Haines, J.L., 2009. Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25. 1. Nat. Genet. 41, 324–328. Zuppan, P., Hall, J., Lee, M., Ponglikitmongkol, M., King, M.-C., 1991. Possible linkage of the estrogen receptor gene to breast cancer in a family with late-onset disease. Am. J. Hum. Genet. 48, 1065.