|Year : 2017 | Volume
| Issue : 1 | Page : 26-32
The association between rs1972820 and the risk of breast cancer in Isfahan population
Narges Zabihi1, Samira Sadeghi2, Hossein Tabatabaeian2, Kamran Ghaedi3, Mansoureh Azadeh4, Mohammad Fazilati1
1 Department of Biochemistry, Payame Noor University of Isfahan, Isfahan, Iran
2 Department of Biology, Division of Genetics, Faculty of Sciences, University of Isfahan, Isfahan, Iran
3 Department of Biology, Division of Cellular and Molecular Biology, Faculty of Sciences, University of Isfahan; Department of Cellular Biotechnology, Cell Science Research Centre, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
4 Zistfanavari Novin Biotechnology Institute, Isfahan, Iran
|Date of Web Publication||16-May-2017|
Department of Biology, Division of Cellular and Molecular Biology, Faculty of Sciences, University of Isfahan, 81746-73441, Isfahan
Source of Support: None, Conflict of Interest: None
Context: A number of single nucleotide polymorphisms (SNPs) in ERBB4 gene have been linked to increase the risk of breast cancer. However, no study has been dedicated to analyze the significance of microRNA-related SNP rs1972820, located in ERBB4 3'-untranslated region (UTR), in breast tumors.
Aims: Here, we investigated the frequency and association between rs1972820 and breast cancer.
Subjects and Methods: The rs1972820 genotypes in 182 samples were collected from 96 healthy people, and 86 breast cancer patients were determined using tetra-primer amplification refractory mutation system-polymerase chain reaction. The frequency of genotypes was analyzed to find the association between rs1972820 and breast cancer risk.
Statistical Analysis Used: Conditional logistic regression, odds ratios (ORs), the associated 95% confidence intervals (CIs), and Armitage's test were used in this study.
Results: In silico analysis suggested that rs1972820 located in the 3'UTR of ERBB4 gene affects the binding affinity of miR-3144-3p a potential oncomiRNA. Statistical analysis showed a significant association between SNP rs1972820 G allele and reduced breast cancer risk, odds ratio = 0.443 (95% CI: 0.196–0.998).
Conclusions: rs1972820 SNP allele is significantly associated with the reduced risk of breast cancer and could be considered as a potential marker for breast cancer predisposition in population of Isfahan.
Keywords: Breast cancer, ERBB4, microRNA-related single nucleotide polymorphism, rs1972820, tetra-primer amplification refractory mutation system-polymerase chain reaction
|How to cite this article:|
Zabihi N, Sadeghi S, Tabatabaeian H, Ghaedi K, Azadeh M, Fazilati M. The association between rs1972820 and the risk of breast cancer in Isfahan population. J Can Res Ther 2017;13:26-32
|How to cite this URL:|
Zabihi N, Sadeghi S, Tabatabaeian H, Ghaedi K, Azadeh M, Fazilati M. The association between rs1972820 and the risk of breast cancer in Isfahan population. J Can Res Ther [serial online] 2017 [cited 2017 Jun 26];13:26-32. Available from: http://www.cancerjournal.net/text.asp?2017/13/1/26/183202
Narges Zabihi and Samira Sadeghi contributed equally to this work.
| > Introduction|| |
Breast cancer is the first-rank malignancy in women aged 20–59 years. According to breast cancer etiology, hereditary genetic factors are responsible for approximately 5–10% of breast cancer cases, due to germ-line variants in breast cancer risk-increasing genes such as BRCA1 and BRCA2. Despite some reported associations between germline variants and enhanced the risk of breast cancer, little is known about the prognostic importance of hereditary variants. ErbB2 receptor tyrosine kinase 4 or ErbB4 is a member of epidermal growth factor receptor (EGFR) subfamily. This subfamily of receptor tyrosine kinases comprises four members including ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). ErbB4 is generated in at least four different isoforms as a consequence of alternative splicing process  Despite of numerous researches on the molecular biology of ErbB4 in normal mammary tissue and also breast tumors, the carcinogenic significance of ErbB4 in breast tissue is still obscure and poorly understood. Different studies investigating the carcinogenic role of ErbB4 have suggested that ErbB4 expression is typically associated with positivity status of hormone receptors including estrogen receptor (ER) and progesterone receptor (PR), negativity of HER2, well-differentiated phenotype, and propitious outcome.,,, Moreover, normal expression and overexpression of ErbB4 gene have been reported to be associated with shorter relapse-free survival compared with patients with low expression of ErbB4 and adverse clinical outcome in patients with ErbB4 overexpression. Furthermore, growth suppression of breast tumor by ErbB4-targeted monoclonal antibody  has been suggesting that ErbB4 could have an oncogenic function in breast cancer. On the other hand, other experiments have shown that ErbB4 activation causes differentiation. For instance, in breast cancer cell lines, it induces the expression of milk proteins and fat droplets. Some studies have reported a loss of ErbB4 expression in breast cancer, and also showed that the expression of ERBB4 gene is lost during breast tumor progression. By generating a constitutively active ERBB4 allele, it has been showed that activated ERBB4 gene is associated with the enhanced cell-killing activity in breast cancer cells. Furthermore, upregulation of ErbB4 protein in ErbB4-expressing breast cancer cell lines resulted in cell cycle arrest and induced differentiation. Moreover, treatment with heregulin, an ErbB4 ligand, induced BRCA1 activity and suppressed cell proliferation regarding both arresting cell cycle in G2-M phase and stimulation of apoptosis only in ErbB4-positive breast cancer cells., These findings, altogether, suggest that ErbB4 could play a role as a tumor suppressor in cells.
microRNAs (miRNAs) are small noncoding RNAs, constituting of 18–24 nucleotides and are endogenously and functionally expressed in eukaryotic cells. miRNAs can negatively regulate protein expression via directly binding to their target mRNAs, typically to mRNA 3'-untranslated region (3'UTR)., miRNAs are initially transcribed by RNA Polymerase II in the nucleus as primary miRNA and then, they are trimmed to ~75-nucleotide precursor miRNA (Pre-miRNA) by the enzyme Drosha. Translocating to the cytoplasm, Pre-miRNAs are processed to mature miRNA by the enzyme Dicer. Mature miRNAs can take action posttranscriptionally as a member of RNA-induced silencing complex, by complementary sequence-specific base pairing to target mRNAs. Abnormal expression or dysregulation of miRNAs has been associated to many types of cancer. miRNAs mainly bind to their target mRNAs through sequence-specific interactions between the miRNA 5' ends (seed region) and 3'UTRs of the target transcripts. Genetic alterations in the 3'UTR of targeted mRNAs have been reported to change the strength of miRNA-mRNA binding, by either gaining or losing the affinity between mRNA and miRNA. These alterations can affect the regulation of target genes; therefore, they might be associated with risk of cancer.,,
The role of variations in different parts of ERBB4 gene has not been extensively studied in breast cancer. In a genome-wide association study (GWAS) carried out in the Korean population, ERBB4 single nucleotide polymorphism (SNP) rs13393577 depicted an association with breast cancer risk. Three ERBB4 risk variants including rs7558615, rs7564590, and rs905883 have also been discovered in a family-based GWAS study. In 2011, the SNP rs1595066, located on the miRNA-binding site of ErbB4 transcript, was reported to be associated with the reduction risk of breast malignancy. However, other miRNA-binding sites located on ERBB4 3'UTR region have not been studied to evaluate the potential diagnostic or prognostic roles of ERBB4 miRNA-related SNPs.
Based on in silico analysis, we identified a miRNA-related SNP rs1972820 present in all four isoforms of ERBB4 and located within the miRNA-3144-3p target site in ERBB4 3'UTR. The aims of this study were to analyze the frequency of SNP rs1972820 which is located on the miRNA-binding site of ERBB4 3'UTR, using tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method , and also to evaluate the diagnostic and prognostic values of this variant. The results indicated that rs1972820 was significantly associated with breast cancer risk.
| > Subjects and Methods|| |
Peripheral blood samples were collected from 86 women breast cancer patients and 96 healthy blood donors as the normal (control) cases. The Ethics approval was performed based on the criteria of the Iranian Ministry of Health and Medical Education that was considered when this proposal approved by the Advisory Board of Payame Noor University of Isfahan. Blood donors were all between 25 and 85 years old. Normal specimens were collected randomly from female subjects attending for regular health examination. Important clinical data obtained from patients is listed in [Table 1].
DNA was extracted from peripheral blood samples using PrimePrep Genomic DNA Isolation Kit from Blood (GeNetBio, Korea), according to the manufacture's instruction. The isolated DNA was diluted in 0.5 M TE and then stored at −20°C.
The 5'- and 3'-flanking sequences of rs1972820 were initially taken from the National Center for Biotechnology Information (NCBI) build 37.3 databases. Tetra-primer ARMS-PCR primers were then designed using PRIMER1 web-based software (http://primer1.soton.ac.uk/primer1.html). All primers were accurately checked by Oligo 7 software package (Molecular Biology Insights, Inc. DBA Oligo, Inc.) to alleviate primer-dimer formations. In tetra-primer ARMS-PCR technique, two sets of primers (outer and inner) are required for SNP and wild-type genotyping. The outer primer set comprises outer forward and outer reverse primers, and the inner set comprises inner forward and inner reverse primers. The sequence of primers is listed in [Table 2].
|Table 2: Tetra-primer amplification refractory mutation system-polymerase chain reaction primers|
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Tetra-primer amplification refractory mutation system-polymerase chain reaction and genotyping
Tetra-primer ARMS-PCR reaction was performed in a final volume of 25 μl, including 100 ng genomic DNA, 3.5 μl × 10 solution buffer, 1.5 μl mixed dNTPs (10 mM), 1.5 μl MgCl2(100 mM), 0.25 μl 5 μ/μl Taq DNA polymerase (Bioron, Germany), and appropriate concentrations of each primer. Temperatures as well as the MgCl2 concentration gradients were exploited for optimizing multiplex PCR conditions. The optimized amplification conditions of the experiments included the initial melting step of 95°C for 5 min, 35 cycles of denaturation in 95°C for 30 s, annealing temperature of 65°C for 40 s, the extension period in 72°C for 50 s, and final extension in 72°C for 10 min. The PCR products length for control, wild-type, and SNP bands were 469bp, 183bp, and 340bp, respectively. The PCR products were separated using 2% agarose gel electrophoresis and ethidium bromide staining. Fifteen DNA samples of different genotyping results including 5 wild-type homozygotes, 5 heterozygotes, and 5 SNP homozygotes were randomly sent to Bioneer (Korea) for Sanger sequencing, as the gold standard method, using ABI 3730XL DNA Analyzer. All sequencing results were analyzed by Chromas Lite software (Version 2.0, Technelysium Pty. Ltd., Tewantin, Queensland, Australia).
Conditional logistic regression, odds ratios (ORs), the associated 95% confidence intervals (CIs), and Armitage's test were executed using DeFinetti program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) to analyze the association between rs1972820 and breast cancer and also an association between rs1972820 and histopathological features of patients. P< 0.05 was considered significant.
MicroRNAs-single nucleotide polymorphism interaction analyses
miRNASNP  Database version 2.0 was used to identify the miRNAs with ability to bind to the 3'UTR of ERBB4 gene transcript. We also used this database to predict the effect of rs1972820 in modifying the binding affinity between miRNAs and ERBB4 mRNA (gain/loss) as well as the changes in Gibbs free energy (ΔG) of this binding.
Single nucleotide polymorphism data
We obtained information of rs1972820 including its minor allele frequency and SNP 5'- and 3'-flanking sequences from NCBI build 37.3 database, and got SNP location from 1000 genome project.
Signal transduction enrichment analysis
Using miRDB, TargetScan (release 6.2, http://www.targetscan.org/), and RNA22 version 2.0 databases, we obtained the predictive targetome of rs1972820-related miRNAs. In the next step, the database for annotation, visualization and integrated discovery (DAVID) database version 6.7, was recruited for signaling pathway enrichment analysis. It helps to identify the statistically significant signaling pathways related to the obtained rs1972820-related miRNAs.
| > Results|| |
Optimization of tetra-primer-amplification refractory mutation system multiplex polymerase chain reaction conditions
The temperature gradient of 55–65°C and the concentration gradient of MgCl2 from 2 to 4 mM were performed for each set of primers in separate vials. The optimized condition to perform PCR in the separate vials was 62°C for annealing step and 6 mM MgCl2. Control, wild-type allele, and SNP allele products related to different genotypes and with optimal annealing temperature and MgCl2 concentration are shown in [Figure 1]a. To achieve the optimum condition of multiplex PCR, i.e., performing all three possible reactions in only one vial, specific concentrations of primers as well as the increased level of MgCl2, dNTP, and PCR Buffer were incorporated. Different ratios of inner and outer primers depicted the optimized condition, in which, it was 3 pM and 6 pM for outer and inner primers, respectively [Figure 1]b.
|Figure 1: (a) Optimization of tetra-primer amplification refractory mutation system-polymerase chain reaction. Lanes 2, 3, and 4 indicate the heterozygote, lanes 5, 6, and 7 wild-type homozygote (A allele) and 8, 9, and 10 single nucleotide polymorphism homozygote (G allele) genotypes. Each genotype was determined in 3 vials. (b) Optimization of multiples tetra-primer amplification refractory mutation system-polymerase chain reaction. Lanes 12–14 show the heterozygote, wild-type homozygote (A allele), and single nucleotide polymorphism homozygote (G allele), respectively. Each genotype was determined in multiplex manner. Lanes 1, 11, and 15 display the 50 bp DNA ladder. The polymerase chain reaction products length for control, wild type, and single nucleotide polymorphism bands were 469bp, 183bp, and 340bp, respectively|
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Frequency of rs1972820 variation
Genotyping of 86 breast cancer patients and 96 healthy cases showed the presence of 42 SNP (G) alleles (G allele frequency ~12%) including 22 heterozygotes and 10 homozygotes in the studied population. The frequency of A/G alleles and different genotypes are shown in [Table 3] and [Table 4], respectively. To test the reliability of tetra-primer ARMS-PCR results, 15 DNA samples of different genotyping results including 5 wild-type homozygotes, 5 heterozygotes, and 5 SNP homozygotes were randomly sent for genotyping by sequencing technique, as the gold standard method. The sequencing outcomes of all 15 specimens were consistent with our results, showing the reliability and accuracy of our optimized tetra-primer ARMS-PCR genotyping method.
|Table 4: Frequency of different genotypes of single nucleotide polymorphisms rs1972820|
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Statistical analysis showed a significant association between G allele and breast cancer (P < 0.05) with odds ratio = 0.443 (95% CI: 0.196–0.998). It means that breast cancer patients were carriers of G allele less often than expected, in comparison with A allele carriers. However, the common odds ratio of GG and AG genotypes, calculated by Cochran-Armitage trend test, was not statistically significant (P > 0.05), reflecting no significant association between different genotypes of SNP rs1972820 (homo and heterozygotes) and breast cancer. Moreover, no significant association between rs1972820 and histopathological features of breast malignancy including positivity of ER/PR/HER2 receptors, lymph node positivity, and grade of cancer was observed (Fisher's exact test, P< 0.05) [Table 5].
|Table 5: Distribution of singlenucleotide polymorphisms rs1972820 G allele and histopathological characteristics of studied breast cancer patients|
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Effect of single nucleotide polymorphism rs1972820 in ERBB4 3' untranslated region -binding microRNAs
Based on miRNASNP database results, a single nucleotide substitution in 3'UTR of ERBB4 transcripts might alter the binding affinity of miR-3144-3p. An A to G substitution (which is U to C in mRNA) might break a normally-present hydrogen bond between ERBB4 mRNA 3'UTR and the seed region of miR-3144-3p. This probable alteration could increase the binding reaction ΔG from −25.50 (stable reaction) to 0 (unstable reaction) [Figure 2]; therefore, G allele (minor allele) could result in upregulation of ERBB4 gene, by increasing the binding affinity of miR-3144-3p to the 3'UTR of ERBB4 mRNA. However, biochemical tests are required to be performed to validate this hypothesis.
|Figure 2: miR-3144-3p binding affinity to its complementary sequence on ERBB4 3' untranslated region, in terms of different genotypes of single nucleotide polymorphism rs1972820. A single nucleotide substitution in 3' untranslated region of ERBB4 transcripts can alter the binding affinity of miR-3144-3p. An A to G substitution (which is U to C in mRNA) can break a normally-present hydrogen bond between ERBB4 mRNA 3' untranslated region and the seed region of miR-3144-3p. This alteration increases the binding reaction ΔG from −25.50 (stable reaction) to 0 (unstable reaction)|
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Molecular signaling pathway enrichment analysis of miR-3144-3p targetome suggests possible role of miR-3144-3p in cancer signaling pathways
To understand how possibly miR-3144-3p is related to cancer, a molecular signaling pathway enrichment analysis was performed. Using miRDB, TargetScan, and RNA22 database, the predicted targetome of miR-3144-3p was obtained and used for further molecular enrichment analysis. Imputing Ensembl gene IDs of miR-3144-3p targetome into a functional annotation tool of DAVID database showed statistically significant related KEGG signaling pathways [Table 6], in which, the most important ones in cancer pathways include MAPK, ErbB, VEGF, p53, and notch signaling pathways. Interestingly, consistent with miRNASNP database, ErbB4 is introduced as a putative target of miR-3144-3p, indicating the reliability of interaction between ErbB4 3'UTR and miR-3144-3p seed region.
| > Discussion|| |
Although clinical investigations on the association of ErbB4 expression pattern with the survival of breast cancer patients are still contradictory,,,,,, in vitro studies as well as mouse xenograft data propose an oncogenic activity of ErbB4 protein in breast tumors ,, while some other studies have introduced ERBB4 as a tumor suppressor gene.,, Some population-based studies benefiting from GWAS technique have reported germ-line ERBB4 variants associated with breast cancer risk, including rs13393577, rs905883, rs7558615, and rs7564590.
More recently, some studies have been conducted to analyze the association between miRNA-related SNPs in 3'UTR of target genes and breast cancer risk. miRNAs are small noncoding RNAs with approximate 22 nucleotides which can negatively regulate protein expression via directly binding to their target mRNAs., miRNAs mainly bind to their target mRNAs through sequence-specific interactions between the miRNA 5' ends (seed region) and 3'UTRs of the target transcripts. Genetic alterations in the 3'UTR of targeted mRNAs have been reported to change the strength of miRNA-mRNA binding, by either gaining or losing the affinity between mRNA and miRNA. These alterations can affect the regulation of target genes; therefore, they might be associated with risk of cancer.,, The genotype CC of rs16917496 in 3'UTR of SET8 has been reported to be associated with earlier age of breast cancer onset compared with TT genotype in Asian patients. Analyzing SNP rs115160714, within 3'UTR of TOPBP1, in Caucasian patients indicated that CT and TT genotypes had significantly elevated risk for breast cancer in comparison with common genotype (CC). In 2011, rs1044129 within the 3'-UTR of RYR3 was reported to be associated with breast cancer risk. In 2008, Brendle et al. showed that there is a strong association between the rare A allele of the SNP rs743554 located within 3'UTR of ITGB4 gene and the risk for ER-negative breast carcinomas. The heterozygote (AC) and homozygote (CC) genotypes of MDM4 rs4245739 and the C variant allele of SNP rs7963551 located in 3'-UTR of RAD52 have been reported to be significantly associated with decreased breast cancer risk., In 2009, and by analyzing rs2747648 within ESR gene, a significant association was reported for the T allele among premenopausal women.
The only study devoted to analyze miRNA-related SNPs in ERBB4 in breast cancer was carried out in 2011, by which it was shown that the SNP rs1595066, located on the miRNA-binding site of ErbB4 transcript, is associated with the reduction risk of breast malignancy. However, other miRNA-binding sites located on ERBB4 3'UTR region have not been studied to evaluate the potential diagnostic or prognostic roles of ERBB4 miRNA-related SNPs. Here, for the first time and to the best of our knowledge, we analyzed the frequency and the diagnostic and prognostic significance of ERBB4 variant rs1972820 in a group of 86 breast cancer patients and 96 healthy (control) cases.
The frequency of the SNP rs1972820 allele was around 8% of patients (14 out of 172 alleles) and 15% in control cases (28 out of 192 alleles), respectively. The frequency of AA (wild-type homozygote), GA (heterozygote), and GG (wild type homozygote) genotypes was 88%, 7%, and 5%, respectively on breast cancer patients while it was 77%, 17%, and 6% in control cases. These data showed a significant association between G allele and reduced risk of breast cancer.
According to RNA-Seq data, miR3144-3p is overexpressed in liver cirrhosis and hepatocellular carcinoma  and colorectal cancer. Therefore, miR-3144-3p is proposed to be a kind of oncomiRNA which eventually results in tumorigenesis. The association between rs1972820 G allele and reduced risk of breast cancer is more compatible with the role of ERBB4 gene as a tumor suppressor. Indeed, as rs1972820 G allele decreases the binding affinity of miR-3144-3p (an oncomiRNA) to ERBB4 3'UTR, it might cause an increase in expression level of ERBB4 (as a tumor suppressor) which, in turns, reduces the risk of breast cancer. However, this assumption requires further investigations to be evaluated.
Among various techniques used for SNP genotyping such as ARMS, RFLP-PCR, and MTPA-PCR, tetra-primer ARMS-PCR technique was performed in this study due to its simplicity, efficiency, and cost-effectiveness advantages. This is the first indication of SNP rs1972820 prognostic significance which is located within 3'UTR of ERBB4 in breast cancer. However, these findings should be validated in a larger population of patients and controls. It is also suggested that other ERBB4 miRNA-related SNPs be encompassed in a study. To confirm the bioinformatic-based predictions, it is strongly recommended to perform luciferase assay to evaluate the changes in gain of binding of predicted miRNA (miR-1344-3p) to the complementary region with both wild-type and SNP sequences.
This study shows that rs1972820 SNP allele is significantly associated with the reduced risk of breast cancer, at least in part through miR-1344-3p-mediated regulation, which may be involved in the pathogenesis of breast cancer. This report proposes a hypothesis that rs1972820 might be used as a potential marker for breast cancer predisposition in population of Isfahan.
| > Conclusions|| |
rs1972820 SNP allele is significantly associated with the reduced risk of breast cancer and could be considered as a potential marker for breast cancer predisposition in population of Isfahan.
We strongly appreciate the staffs of Seyed-al-Shohada Hospital and our colleagues in ZistFanavari Novin Biotechnology Institute for their constructive participations and comments.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]