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Introduction: In women, breast cancer is a prevalent malignancy. One of the important genes in breast cancer progression is Pinin. LncRNAs H19 plays an important role in breast cancer cell development. In this study, the correlation between a miRNA-related rs532 polymorphism (G) and the level of expression of Pinin and lncRNA H19 in breast cancer tissues was investigated. Methods: Tissue samples were collected from 64 patients with breast cancer. RT-PCR with highresolution melting (HRM) analysis was used. Result: Our data suggest that there is significant up-regulation in the expression of lncRNA H19 and the Pinin gene in breast cancer tissue compared to the control ones. The Pinin polymorphism rs532 G is significantly associated with Pinin expression of breast cancer tissue compared to the control one. Discussion: The rs532 G polymorphism is located around the pinin and its allele frequency is significantly associated with Pinin expression. Perhaps, rs532 G polymorphism could be functional in breast cancer progression through the impact on pinin expression. There is no correlation between SNP and lncRNA H19. Conclusion: The polymorphism rs532 G polymorphism is significantly associated with Pinin expression of breast cancer tissue from Isfahan patients which suggests the miRNA-141has potential to regulate Pinin expression levels.

Treating breast cancer is hardly achieved and finding efficient anticancer nanocompounds has gained great attention. In this work, the cytotoxic effect of GaFe2O4@Ag nanocomposite biosynthesized by Spirulina platensis on breast cancer cell line and expression of the BAX, Bcl-2, CASP9 and MMP1 (Matrix Metallopeptidase 1) genes were evaluated. Physicochemical features of the nanocomposite were determined using the FT-IR, XRD, SEM, TEM, EDX-mapping, VSM, Zeta potential, and DLS analyses. The cytotoxic effect of the nanocomposite for MCF-7 and HEK-293 cells was evaluated by the MTT assay. Flow cytometry analysis, Caspase-3 activation assay, and Hoechst staining were performed to evaluate the apoptosis induction potential of the nanocomposite. Further, the relative expression of the Bcl-2, BAX, and CASP9 genes was determined by quantitative PCR assay. The prepared nanocomposite was spherical with a size range of 35–60 nm. The hydrodynamic size and zeta potential of the nanocomposite were 328 nm and − 31.8 mv, respectively. GaFe2O4@Ag nanocomposite had a higher cytotoxic effect on breast cancer cells than normal human cells with the IC50 of 18.6 and 220 µg/mL, respectively. Treating breast cancer cells with the nanocomposite induced apoptosis among 85.2% of cells, increased caspase-3 activity by 4.3 folds, and caused apoptotic nuclear changes. Also, GaFe2O4@Ag reduced the expression of the Bcl-2 and MMP1 by 1.3 and 0.6 folds and up-regulated the BAX and CASP9 genes by 2.7 and 2.65 folds, respectively. Our results revealed that GaFe2O4@Ag was highly cytotoxic for breast cancer cells via triggering apoptosis pathways and could be considered as a novel and efficient agent against breast cancer, after further in-vivo experiments.

Background: Among women worldwide, breast cancer is the most common form of cancer. Malignant tumors are believed to be regulated by long noncoding RNAs (lncRNAs). By using both experimental and bioinformatic approaches, we examined the expression level of ZNF667-AS1 and its interactions with correlated microRNAs, lncRNAs, and mRNAs or protein-coding genes. Material and methods: An RNA interaction analysis (miRWalk, lncRRisearch, ENCORI, and lncBase3) was performed along with a protein interaction analysis (STRING). As part of this study, we analyzed the differentially expressed genes inthe microarray dataset GSE61304, as well as in breast cancer tissue samples,using quantitative real-time PCR. Result:Significantly low expression of ZNF667-AS1 was found in the ENCORI experiment (change: 0.61, FDR < 0.0001) and qRT-PCR experiment (fold change: 0.046071, p value < 0.0001). The ROC curve indicates that ZNF667-AS1 has potential as a diagnostic biomarker of breast cancer(AUC of 0.8373, p value Conclusion: ZNF667-As1 was found to be a promising diagnostic biomarker for breast cancer samples based on this integrated computational and experimental study. In the future, researchers will be able to gain more insight into the role ZNF667-AS1 plays in breast cancer by investigating the correlation between ZNF667-AS1 and correlated mRNAs, lncRNAs, and microRNAs, especially the RNAs mentioned in this study.

Background Breast cancer (BC), one of the ten most common diseases in the world, is a common malignant tumor that forms from the women's breast epithelial tissue. According to Cancer Statistics 2020, over 1.7 million new cancer cases are detected yearly. In this research, we aimed to evaluate the expression level and interaction network of the SP1 gene, and it’s regulatory non-coding RNAs in the BC samples. Method High-throughput microarray data analysis was performed on GSE42568 (27 control and 31 tumors) to find the up and down-regulated genes in BC patients. Reactome performed pathway enrichment analysis. Enrichr performed gene ontology analysis. Comprehensive protein-protein (STRING), miRNA-mRNA (miRWalk), and miRNA-lncRNA (lncBase 3) was performed to find the RNA regulatory network of SP1. Cytoscape performed visualization of RNA network. The qRT-PCR experiment was performed to validate the changes in the expression level of SP1 and related non-coding RNAs in the BC samples. Results Microarray analysis revealed that SP1 has a significantly low expression in the BC samples. lncRNA HCG11 regulates SP1 in direct interaction and ceRNA axis (binding affinity to hsa-miR-106a-3p). hsa-miR-106a-3p regulates the expression level of SP1 during regulating the transcriptional activity of the SMAD2/SMAD3:SMAD4 heterotrimer process. qRT-PCR experiment revealed that SP1 (logFC: -4.174, p-value: 0.0007) and lncRNA HCG11 (logFC: -4.223, p-value: 0.0003) have significant low-expression in the BC samples. ROC analysis revealed that SP1 (AUC: 0.7312, p-value: 0.07074) HCG11 (logFC: 0.7312, p-value: 0.07505) could be the two potential diagnostic biomarkers of BC. Conclusion SP1 and lncRNA HCG11 have significantly low expression in the BC samples as the two potential diagnostic biomarkers. Has-miR-106a-3p regulates the expression level of HCG11 and SP1 in a ceRNA axis. This axis might be a novel regulatory factor in response to the hydroperoxide process.

IgE levels increase in some cancer types due to treatment methods also autoimmune diseases can emerge and lead to worsening of the cancer. Finding a factor based on the reduction in IgE levels found in cancer can act as a determinant in providing the early diagnosis of the disease in individuals with high IgE levels in their blood. Accordingly, in the present study, the effort was made to select a gene associated with breast cancer and high IgE levels through available databases to evaluate the gene expression and the associated lncRNA and polymorphism at the binding site of a miRNA with a gene. In this study, the expression of the VTCN1 gene and the associated lncRNA called NORAD (Non-Coding RNA Activated by DNA Damage), selected through bioinformatics methods and relevant databases were assessed in breast cancer tissue samples along with the blood samples of individuals with high IgE levels, employing the real-time PCR method. Lastly, NORAD’s protein interactions and the relevant signaling pathways were further investigated via bioinformatics tools. The results indicated that the expression of VTCN1 genes increased significantly (P NORAD increased insignificantly (P > 0.05) in both breast cancer and high IgE level samples. The prevalence of GG genotype in polymorphism rs10754339 was similarly high in both categories of samples. Bioinformatic analyses also demonstrated NORAD's association with proteins involved in immunity pathways. This study suggests the VTCN1 and NORAD genes as convenient markers for monitoring breast cancer in people with high IgE levels, as well as employing them in the research regarding the autoimmune diseases in individuals with a breast cancer diagnosis.

Breast cancer is considered a life-threatening disease among females globally which early diagnosis and treatment in the initial stages can be very effective. SMAD4 is a key regulator of TGF-β pathway that is known to play an essential role in breast cancer. Long non-coding RNAs (lncRNAs) are considered to be potential regulatory factors for several cancers that are not definite about the main role of two LncRNAs, LINC00909 and LINC00667, on breast cancer. In this study, we focus on identifying the possible correlation between expression levels of SMAD4 gene, LINC00909 and LINC00667 in breast carcinoma, as well as their potential roles in the diagnosis of breast cancer. To determine the extent of expression of these factors in 25 paired tumor-normal patient tissues, quantitative real-time PCR (polymerase chain reaction) was used. Statistical analysis showed a significant increase in the expression of the SMAD4 gene in tumor cells compared with control samples, while the expressions of LINC00909 and LINC00667 increased dramatically. As well as, specificity and sensitivity between all three criteria and breast carcinoma were statistically significant. furthermore, we observed the positive relationship between expression of LINC00909 and HER2/neu in the tumor tissues, whereas there was not another correlation between clinicopathological features and SMAD4 gene, LINC00909 and LINC00667. Moreover, we discovered a positive association between the expression of LINC00667 and LINC00909. altogether, we conclude that, SMAD4 could be considered as a potential diagnostic biomarker for breast cancer, and two lncRNAs, especially LINC00909, were identified to play an important role in the identification of breast cancer.

Background Gastric cancer (GC) is the second most frequent cause of cancer-related death worldwide and the fourth most common malignancy. Despite significant improvements in patient survival over the past few decades, the prognosis for patients with GC remains dismal because of the high recurrence rate. In this comprehensive systems biology and experimental investigation, we aimed to find new novel diagnostic biomarkers of GC through a regulatory RNA interaction network. Methods Gene expression, co-expression, and survival analyses were performed using microarray and RNAseq datasets (analyzed by R Studio, GEPIA2, and ENCORI). RNA interaction analysis was performed using miRWalk and ENCORI online databases. Gene set enrichment analysis (GSEA) was performed to find related signaling pathways of up and down-regulated genes in the microarray dataset. Gene ontology and pathway enrichment analysis were performed by the enrichr database. Protein interaction analysis was performed by STRING online database. Validation of expression and co-expression analyses was performed using a qRT-PCR experiment. Results Based on bioinformatics analyses, THBS2 (FC: 7.14, FDR

Background: One of the most common female malignancies is breast cancer (BC) and is recognized as a second death factor for women population among other cancer-related diseases. Dysregulation of RNA expression levels can lead human status to some different pathological statuses, including breast cancer. In this study, we have investigated the expression level of DENND2A and lncRNA LINC02544 in the BC tissue samples among Iranian population. Also, the clinicopathological and biomarker analysis of these RNAs were investigated. Method: Microarray analysis was performed on GSE61304 in order to find the differentially expressed genes in the BC samples compared to controls. Multiple online and offline software, including R programming language and several relative statistical and visualization packages, demonstrated the differential expression level of genes,relative lncRNAs, and single nucleotide polymorphisms. The Real-time PCR results have demonstrated that the expression level of DENND2A has been reduced in the breast cancer samples compared with controls. In order to investigate the genotype frequency of rs6852 region in the DENND2A gene, high-resolution melt (HRM) method has been used. Results of microarray and Real-time PCR analysis have been shown that the expression of DENND2A and lncRNA LINC02544 have significantly down-regulated in the breast cancer samples. These data suggested that these two RNAs are likely to respond to alter the expression level of each other. Therefore, DENND2A could be an important prognostic biomarker in breast cancer samples among Iranian population. lncRNA LINC02544 could have an activator effect on the DENND2A expression. There is strong evidence that both of these RNAs could perform as tumor suppressors in breast cancer.

long non-coding RNAs play essential roles in the regulation of the gene’s expression level. The abnormal difference in the gene expression and transcriptome amount in the cells can make the various diseases in the human, including cancer. In this study, the expression of MYC and the two relevant and co-expressed lncRNAs were analyzed in the breast cancer (BC) samples as the potential BC biomarkers. An integrated bioinformatics analysis – including Microarray, RNA interaction, Pathway enrichment, and Gene ontology analyses – was performed to find novel differentially expressed genes in the BC patients. A real-time PCR experiment evaluated the expression of potential BC biomarkers found in the bioinformatics analyses. Bioinformatics and experimental analyses revealed that MINCR and JPX have a remarkable up-regulation in the BC samples and can be the two BC oncogene. Also, it is demonstrated that MYC could act as a tumor suppressor in BC patients by low-expression in the BC samples. All in all, the changes in the expression of MYC – affected by MINCR and JPX – can promote breast cancer pathogenicity. These three coding and non-coding RNAs can act as the acceptable prognostic biomarkers in BC.

Background The primary goal of this study is to compare the levels of expression of ID1, LINC00101, and LINC00652 in Isfahan Breast cancer samples to control samples. Understanding the relationship between variations in RNA expression levels and the clinicopathological statuses of Isfahan patients is another important goal of this research. Method R Studio evaluated 104 breast cancer tissue and 17 control samples in the GSE42568 microarray datasets to identify a relevant dysregulated gene. The limma package was used to conduct differential expression analysis. The DEGs were used to test mRNA-lncRNA interactions using the co-lncRNA online database. The qRT-PCR experiment aimed to determine the degree of expression of ID1, LINC001013, and LINC00652. Result Based on bioinformatics and experimental analyses, ID1 has a significant low-expression in the BC samples. Furthermore, lncRNAs LINC00101 and LINC00652 have significant high expression in the BC samples. ID1 and LINC00652 might be the two potential diagnostic (ROC analysis) and prognostic (Clinicopathological analysis) biomarkers of BC. Discussion According to ROC and clinicopathological analyses, ID1 and LINC00652 could be the two significant prognosis and diagnosis biomarkers in Isfahan Breast cancer patients.

In addition to coding region, non-coding genes can also be involved in the development of cancer cells. Therefore, the present study aimed to study the expression level of the coding gene PEAR1 and non-coding gene LINC00271 in breast tumors. Using bioinformatics approaches, the DE genes and miRNAs in breast cancer tissue were extracted from datasets. MiRNA-target regulatory network visualization in Cytoscape along with applying Cytohubba lead to identifying hub-miRNAs and their targets in breast tumors. Functional enrichment analysis was done using MIEAA database. The co_lncRNA and lncBASE databases were utilized to find effective lncRNA. Verifying the PEAR1 and LINC00271 expression in breast tumor and adjacent healthy cells was carried by Real-Time PCR assay. Analysis of array-based non-coding expression profile resulted in 96 significant DE miRNAs which 15 out of them were collected as hub-miRNAs due to the number of their target genes. 4 common genes were identified between targets of hub-miRNAs and DE genes in breast tumors; among them, PEAR1 was considered as novel DE gene which also was one of the targets of upregulated hub-miRNAs, hsa-miR-34a-5p. LINC00271 was selected as correlated lncRNA with PEAR1 and hsa-miR-34a-5p. qRT-PCR results revealed that the expression level of both PEAR1 and LINC00271 were significantly down-regulated (Fold change PEAR1 = 0.00798/P-Value P-Value = 0.0094) in breast tumors compared to controls. Due to a significant reduction in PEAR1 and LINC00271 expression and array-based reported, the association between these genetic factors can be considered as new molecular markers in the prognosis and metastasis of breast cancer.

Objectives The transmembrane protein PERP is present in numerous epithelial and epithelium-derived cells in tessellating junctions and other junctional and non-junctional plasma membrane regions. Previous research has focused on the involvement of the PERP gene and associated non-coding RNA regulators in developing several cancer types. In this study, we performed a high-throughput microarray data analysis and related interaction and pathway analysis to find novel genes in breast cancer and perform a qRT-PCR experiment to measure the accurate expression level of genes. Materials and method Microarray data analysis and gene set enrichment analysis (GSEA) were performed to find novel dysregulated genes in the breast cancer (BC) samples. Protein-protein interaction, pathway enrichment, gene ontology (GO), and RNA interaction analyses were performed to find an integrated regulation network. The qRT-PCR experiment was performed to evaluate the expression of found genes. Results According to the bioinformatics analyses, PERP, lncRNA CASC15, and LINC01010 were selected as the hub dysregulated genes in the BC samples, regulating the p53 signaling pathway. These genes were selected from a ceRNA network. PERP (logFC: 2.631, p-value: 0.006), lncRNA CASC15 (logFC: 2.627, p-value: 0.0018), and LINC01010 (logFC: 1.838, p-value: 0.0049) have up-regulation significantly in the BC samples. Also, CASC15 can be considered as a novel diagnostic biomarker of Isfahan BC (AUC: 0.7008, p-value: 0.0149). There were significant correlations between the expression of PERP and LINC01010 and age, menopausal and HER2 receptor statuses of Isfahan patients. Conclusion LINC01010 and CASC15 can regulate the expression level of PERP in the BC patients and change the normal regulation of the p53 signaling pathway and affect the apoptosis process of normal cells. CASC15 as a potential diagnostic biomarker and LINC01010 as a potential prognostic biomarker could regulate the BC development in the different ages, menopausal statuses, and HER2 receptor negative patients.

Background: NME1 is a crucial gene in the proliferation, cancer survival, and differentiation processes. The abnormal changes in the expression of NME1 could cause disturbance in the mentioned processes and lead the normal cells to different abnormalities, including cancer. Breast cancer is the most frequently diagnosed cancer in women and ranks second among causes for cancer related death in women. In this study, the expression pattern of NME1 and the two lncRNAs that have possible interaction with NME1 (LINC01028 and LINC00528) had been evaluated In the Isfahan breast cancer (BC) patients.Methods and results: Microarray, pathway enrichment, Gene Ontology, RNA and Protein interaction, and co-expression analyses were performed to find novel integrated genes, proteins, and RNAs (coding and non-coding) in the BC patients, based on the bioinformatics tools (R programming language and online databases). After finding novel genes and RNAs, a real-time PCR experiment was performed to evaluate the accurate relative expression of found RNAs in the Isfahan human breast cancer samples. Bioinformatics analyses revealed that NME1 protein could regulate apoptotic-related proteins. Also, based on ENCORI and microarray data analysis, the expression of NME1 and LINC00511 (FC: 6.49, FDR < 0.0001) have a significant up-regulation in the BC samples. Also, LINC01028 had a significantly low expression in the mentioned datasets (FC: 0.08, FDR < 0.0001). A real-time PCR experiment validated the bioinformatics results. Based on the experiment, NME1 (logFC: 2.664, P. Value < 0.0001) and LINC00511 (logFC: 2.058, P. Value: 0.0180). Also, LINC01028 have significant low-expression in the BC samples (logFC: -2.219, P. Value: 0.0006).Conclusion: Based on the bioinformatics and experimental analyses, NME1, LINC00511, and LINC01028 could be three prognostic biomarkers in the Isfahan BC population. The disturbance of the expression level of mentioned RNAs can change the regular apoptosis process and promote BC development in the patients.

Background ESR2 can have a remarkable effect on the proliferation and tumorigenesis of breast cancer (BC) as a hub tumor suppressor by regulating the effect of estrogen. The abnormal changes in the expression of the co-expressed lncRNAs can have a regulatory effect on the ESR2 expression and cause BC status. In this research, the expression and correlation of ESR2 and a related lncRNA (LINC00520) were examined through different statistical methods. Method An integrated bioinformatics analysis was performed to obtain a reasonable prediction about the relation of potential biomarkers and design an experiment. Based on the Microarray data analysis, pathway enrichment, GO, and ceRNA interaction analyses, a collection of RNAs was selected for the real-time PCR experiment. Several statistical methods were performed to evaluate the difference in the expressions in the BC samples, correlation of RNAs, and the possibility of being prognostic and diagnostic biomarkers (ROC and clinicopathological analysis). Result It is demonstrated that ESR2 (logFC: -1.842, P. Value LINC00520 (logFC: -2.231, P. Value LINC00520 can significantly help BC development by affecting the expression of ESR2 and playing a tumor suppressor role in Isfahan BC population. Furthermore, LINC00520 could be considered as a significant prognostic (clinicopathological analysis) and diagnostic (ROC analysis) biomarkers of BC.

Background: A disintegrin and metalloproteinases (ADAMs) are a novel gene family of proteins that share the metalloproteinase domain with matrix metalloproteinases and have sequence similarities to the reprolysin family of snake venomases (MMPs). They are divided into two classes based on their structural similarities: membrane-anchored ADAM and ADAM with thrombospondin motifs (ADAMTS). These molecules have a role in a number of biological processes, including membrane protein shedding, proteolysis, cell adhesion, cell fusion, and cell migration. Method: In this study, the RNA interaction analysis (miRWalk, lncRRisearch, ENCORI, and lncBase3) and protein interaction analysis (STRING) was performed. Survival, expression, and correlation analyses was performed by ENCORI and GEPIA2. Expression analysis also performed by microarray analysis. GSEA analysis was performed to find top up and down-regulated genes and relevant signaling pathway. Gene ontology analysis was performed by g: Profiler and clusterprofiler. Pathway enrichment was performed by GSEA analysis.ResultsADAMTS5 has a significant low-expression, based on ENCORI (Fold Change: 0.12, FDR < 0.0001), microarray analysis (logFC: -3.791881, adj. P. Val < 0.0001), and qRT-PCR experiment (logFC: -1.975, FC: 0.025, p. value < 0.0001, patients under 40). ADAMTS5 is a potential diagnostic biomarker of BC, based on ROC curve (AUC: 0.75, p. value: 0.0007). mRNA VTI1B, lncRNAs CRNDE and BAIAP2-AS1, and hsa-miR-135a-3b have interaction with ADAMTS5. VTI1B has a significant low-expression (FC: 0.65, FDR: 0.0001). BAIAP2-AS1 is significantly up-regulated (FC: 1.37, FDR: 0.0001). CRNDE is significantly up-regulated (FC: 1.30, FDR: 0.0001). BAIAP2 (log-Rank p: 0.0017, Hazard Ratio: 0.59) and CRNDE (log-Rank p: 0.34, Hazard Ratio: 1.17) are the two potential BC survival predictor.ConclusionIn this study, we performed a differential expression analysis on the expression level of ADAMTS5, VTI1B, and BAIAP2-AS1 in the BC patients for the first time. ADAMTS5 and its correlated oncogene and tumor suppressor RNAs and proteins are the hub therapeutic target of BC. DEGs of the GSE42568 dataset are involve in PPAR and cell cycle checkpoint signaling pathways and during the regulation of mentioned pathway, regulate the BC development. Targeting the expression and interaction of mentioned RNAs can be helpful for the diagnosis, prognosis, and treatment of BC.

A higher expression of MALAT1 has been reported in breast cancer. However, more studies are needed to decipher the mechanisms by which this lncRNA imposes its oncogenic effects. In this study, blood and tissue samples were taken from healthy normal and breast cancer subjects. qPCR was used to analyze the gene expression. HRM-PCR method was carried out to genotype the selected samples. Computational analysis was recruited to find novel targets of MALAT1 and miR-143-3p. The data analyses revealed that MALAT1 was up-regulated in breast cancer and could be a distinctive factor to diagnose cancer. The expression of MALAT1 was inversely correlated with miR-143-3p expression in the studied clinical samples. The down-regulation of miR-143-3p was proven in the clinical tumor samples as compared to the healthy controls. A negative correlation of miR-143-3p with its putative target, RALGAPA2 was observed. A functional SNP rs3827693 located within the 3'UTR region of RALGAPA2 mRNA was validated in this study to associate with breast cancer risk. The rs3827693 allele G significantly decreased the breast cancer incidence and augmented the negative correlation between RALGAPA2 and miR-143-3p, presumably through strengthening the interaction between these two transcripts. This study proposed MALAT1 miR-143-3p and miR-143-3p RALGAPA2 axis in breast cancer, whereby the latter can be altered by the clinically functional SNP rs3827693.

MUC4 is aberrantly expressed in several carcinomas including breast, colon, ovarian, lung, prostate, stomach and pancreatic cancers. MUC4 can regulate cell apoptosis negatively and facilitate stomach tumorigenesis. In this research, we aimed to evaluate the possible association between rs2641726 (C > A) polymorphism of MUC4 and gastric cancer risk in the Iranian population. In this case-control study, we collected blood samples from 168 gastric cancer patients and 66 healthy subjects. Allele-specific primer polymerase chain reaction method was applied to genotype rs2641726 in the obtained DNA samples. This study demonstrated that rs2641726 C allele was significantly associated with the incidence of gastric cancer, odds ratio = 3.382, 95% confidence interval: 1.840–6.217 (P

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.

Background Gastric cancer is the fifth most common cancer worldwide. Along with environmental factors, such as Helicobacter pylori (H. pylori) infection, genetic changes play important roles in gastric tumor formations. miR-584 is a less well-characterized microRNA (miRNA), with apparent activity in human cancers. However, miR-584 expression pattern in gastric cancer development has remained unclear. This study aims to analyze the expression of miR-584 in gastric cancer samples and investigates the associations between this miRNA and H. pylori infection and clinical characteristics. Methods The expression level of miR-584 was studied in primary gastric cancers versus healthy control gastric mucosa samples using the RT-qPCR method. The clinical data were analyzed statistically in terms of miR-584 expression. In silico studies were employed to study miR-584 more broadly in order to assess its expression and find new potential target genes. Results Both experimental and in silico studies showed up-regulation of miR-584 in patients with gastric cancer. This up-regulation seems to be induced by H. pylori infection since the infected samples showed increased levels of miR-584 expression. Deeper analyses revealed that miR-584 undergoes a dramatic down-regulation in late stages, invasive and lymph node-metastatic gastric tumors. Bioinformatics studies demonstrated that miR-584 has a substantial role in cancer pathways and has the potential to target STAT1 transcripts. Consistent with the inverse correlation between TCGA RNA-seq data of miR-584 and STAT1 transcripts, the qPCR analysis showed a significant negative correlation between these two RNAs in a set of clinical samples. Conclusion miR-584 undergoes up-regulation in the stage of primary tumor formation; however, becomes down-regulated upon the progression of gastric cancer. These findings suggest the potential of miR-584 as a diagnostic or prognostic biomarker in gastric cancer.

Gastric Cancer, is widespread despite medical science progress, and is characterized by a low rate of survival and poor recovery of patients throughout the world. Hence, single nucleotide polymorphisms associated with the binding status of miRNA in the target gene that affects gene expression level in this cancer. The purpose of this study was to investigate the association of rs12977 polymorphism alleles in RAC1 and the prevalence of gastric cancer as well as its progress concerning other clinical and pathological features of Iranian patients. A computational study was initially performed to explore the signaling pathways that correlated with the activity of hsa-miR-3605-3p by way of linking rs12977 was located at its seeding region responsible for the binding with hsa-miR-3605-3p. Real-time PCR-HRM analysis on genomic DNA of stomach cancer (no: 140) and control (no: 54) samples, genotyping approach was carried out to determine the association of rs12977 with the incidence/progress of gastric cancer. Results indicated a significant correlation between polymorphism and primary tumor, the degree of progression of cancer and metastasis (p

Breast cancer is one of the main causes of mortality throughout the world, especially in women. Lysine Demethylase 2A (KDM2A) gene is highly expressed in myoepithelial cells of mammary tissue that eventually contributes to breast tumorigenesis. The purpose of this study was to investigate the association between rs12287003 polymorphic factor of the KDM2A gene and breast cancer incidence. To develop this aim, we used Allele-specific PCR (ASP-PCR) to genotype breast cancer and healthy control samples. The results indicate that rs12287003 allele C was associated with a higher risk of breast cancer, while allele G reduced it. Mechanistically, allele C was shown to broadly attenuate the binding of numerous miRNAs to KDM2A transcripts, potentially inducing the KDM2A upregulation-mediated breast tumorigenesis. The findings of this study may have clinical implications upon validation in other clinical cohorts.

Breast Cancer (BC) is known as common neoplasia mostly in women. Long non-coding RNAs (LncRNAs) are considered as vital effective pathological regulators in cancers. The present study has been done to find the relation between BC and CCAT1 (colon cancer-associated transcript-1 RNA) which is situated in Chromosome 8, as known BC biomarkers. This LncRNA was evaluated in Isfahan, Iran Population. Since it has presented a significant decrease of expression in BC tissue, it may be considered as a great deal of potential to be a biomarker for this population. Quantitative real-time PCR (polymerase chain reaction) was utilized to analyze the expression levels of CCAT1on 74 pairs of tumor-normal breast tissues of BC patients. For statistical analysis, the Genex, R, and Graph prism8 software were used. An Independent student t-test was done to show a significant variation of groups. Based on statistical analysis, the significant tissue down-regulation of CCAT1 in BC patients in comparison to control samples (p = 0.001 respectively) has been identified. R studio examination with t-test indicates that the expression profiles of CCAT1 do not have a significant correlation with stage, lymph-node, tumor size, menopausal ER, PR receptor, and Her2. our Results provide that the downregulation of LncRNA CCAT1 could be considered as a potential diagnostic biomarker for BC.

Background Abnormal gene expression levels of enzymes involved in fatty acid synthesis and oxidation pathways in breast cancer have been shown to associate with progression of breast cancer. However, little is known about the significance of alternations in fatty acid activation, as a key step in their metabolism. We aimed to recognize suitable biomarkers indicating the altered activation of fatty acids and examine their differential expression in breast cancer and adjacent normal breast tissue samples. Material and methods Transcript levels of ACS enzymes were analyzed in TCGA data (RNAseq), breast cancer and normal breast samples by differential gene expression (DGE). Gene set enrichment analysis (GSEA) was performed to highlight the most enriched genes involved in the activation of fatty acids. Bioinformatic analysis was performed for the selection of LncRNAs. Gene expression analyses were performed using quantitative Real-time PCR (qRT-PCR) in breast cancer tissue samples and adjacent normal breast tissue samples. Correlation and survival analyses were performed using R program. ROC curves were created for TCGA and qPCR data to evaluate the prognostic potential of the biomarkers. Results GSEA analysis revealed that ACSM5 is highly enriched in normal breast tissue samples compared to breast cancer tissue samples. LINC00265 with highest co-efficient score with ACSM5 was selected for further analyses. The expression level of ACSM5 and LINC00265 positively correlated with each other and were significantly low in breast cancer tissues compared to normal breast tissues. Correlation analysis suggested a positive correlation between ACSM5 and LINC00265 genes. Survival analysis predicted prognostic potential for ACSM5. ROC curve analyses revealed that ACSM5 and LINC00265 can distinguish breast tumor samples from adjacent breast tissues. Conclusion Our study suggests ACSM5 gene as a potential biomarker with prognostic value in detection of altered activation of medium-chain fatty acids in breast cancer. In addition, the positive correlation between ACSM5 and LINC00265 suggests the presence of possible regulatory mechanism between them.

Background Breast cancer is highly prevalent globally and is one of the main causes of cancer-related deaths worldwide. Despite the advances, more insights are needed to decipher the regulatory mechanisms leading to breast cancer development and progression. Here, we examined the expression of PGR and TUG1 genes in breast cancer. Material and methods qPCR was used to quantitatively measure the expression of PGR and TUG1 transcripts in normal and breast cancer fresh clinical samples. Results The higher expression of both PGR and TUG1 was observed in the breast tumors as compared to the normal adjacent paired samples. The ROC curve analysis revealed that PGR and TUG1 expression patterns can discriminate breast tumor samples against normal tissues. Besides, there was a significant correlation between PGR and TUG1 expression in the studied samples. Conclusion PGR and TUG1 are significantly up-regulated in breast cancer, which both can distinct tumor from normal tissues. With respect to the positive correlation between PGR and TUG1, this gene signature might be used as a diagnostic biomarker for breast cancer detection.

Background Multiple sclerosis (MS) is one of the most prevalent inflammatory and autoimmune diseases, associated with the demyelination of nerve cords. The previous studies have proposed that Tryptophan hydroxylase (TPH) modulation may be associated with neurologic disorders including MS. Thus in the present study, we aimed to investigate whether the presence of two SNPs (rs17110566) and (rs17110747) in the TPH gene could be associated with the risk of MS incidence in the Iranian population or not. Methods In the present case-control study, 210 patients with MS and 153 matched healthy controls were enrolled. Genomic DNA from whole blood was isolated and genotyping was performed using the HRM technique. The data were analyzed by SPSS and p value Results Our results showed a significant difference in allelic frequency of SNP rs17110747 among MS patients and controls (p-value = 0.018). The frequency of AA genotype was evident with an increased risk of MS (p-value = 0.005). Additionally, the AA genotype of rs17110747 increased the risk of onset and expanded disability status scale (EDSS) of MS in females compared to males. Furthermore, there was no significant difference between MS patients and controls in allelic and genotype frequency of SNP rs17110566. Conclusion Taken together, a homozygous type of allele A at rs17110747 in TPH is related to the increased risk of MS development, especially in Iranian women.

Breast cancer has been increasing in developing countries; this cancer is becoming a fundamental health problem for these countries. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic enzyme correlated with the hormonal receptor concentrations, cell proliferation, tumor grade and stage, drug resistance, and outcome of breast cancer cells. Furthermore, genetic variations in miRNA binding sites could be played an essential role in breast cancer risk and survival. In this study, the SNP rs1803622 at the 3′-UTR of GAPDH was chosen as a candidate SNP for investigation based on bioinformatics studies. This polymorphism was genotyped in an Iranian population with 123 breast cancer patients and 117 matched healthy controls using the Allele-Specific Primer (ASP)-PCR method. The functional impact of this genetic variation on the post-transcriptional regulation of GAPDH gene were bioinformatically studied. The genotyping data, for the first time, successfully identified frequencies of GG, GT, and TT to be 7%, 61%, and 32% in breast cancer patients, respectively. In particular, the assessed frequencies of allele G was 38% while risk allele T was 62% in these population. Results revealed that compared with the GG genotype of rs1803622, T allele was related to a significantly increased risk of breast cancer in a dominant genetic model Significant difference in allele and genotype distributions in the SNP rs1803622 was found between the high and low expression levels of estrogen, progesterone, and HER2 receptors. Furthermore, allele and genotype frequencies in the SNP rs1803622 were also related to the stage and grade of breast cancer. Surprisingly, Genotype and allele frequencies in this SNP were also significantly different in metastatic and non-metastatic breast cancer samples. Finally, the bioinformatics data suggested that the SNP rs1803622 might be involved in transcriptional regulation of the GAPDH gene by hsa-miR-548g.

Background Breast cancer is the most common cancer among women worldwide. HER2-positive breast cancer accounts for nearly 20% of all cases. Upon targeted therapy resistance, these patients show fairly poor-prognosis. miRNAs have shown to be differentially expressed in HER2-positive cases that can decipher drug resistance. miR-195 down-regulation has been reported in breast cancer; however, very little is known about its expression particularly in HER2-positive breast cancers. Material and methods qPCR was used to quantitatively measure the expression of miR-195 in normal, HER2-positive and HER2-negative fresh clinical samples. A computational approach was used to find unvalidated but potential targetome of miR-195. Results miR-195 is down-regulated in breast cancer samples as compared to the normal controls. The level of down-regulation was significantly greater in HER2-positive tumors as compared to both normal and HER2-negative samples. Importantly, miR-195 down-regulation was demonstrated to be a potential diagnostic biomarker for distinguishing HER2-positive against both normal and HER2-negative samples. Besides, the in silico approach revealed that unvalidated miR-195 targetome play roles in oncogenic pathways related to breast tumorigenesis and progression. Conclusion miR-195 is down-regulated in breast cancer, especially and significantly in HER2-positive cases. The expression of miR-195 could effectively be used as a distinctive diagnostic marker to discriminate HER2-positive from both normal and HER2-negative samples.

Background: Gastric cancer is one of the most significant reasons for cancer-related death. miR-146a is one of the dysregulated factors associated with gastric tumorigenesis. However, deregulation of this microRNA (miRNA) has become controversial. Moreover, the inflammation-mediating role of this miRNA implies that miR-146a might be dysregulated by gastric cancer-related pathogens, such as Helicobacter pylori. However, the dysregulation of miR-146a in H. pylori-infected gastric tumors has not been widely studied. Objectives: We aimed to analyze the expression level of miR-146a in gastric cancer tissues and then to assess any potential association between miR-146a and H. pylori infection and other clinical characteristics. Materials and Methods: miR-146a expression level was quantitatively studied by reverse transcription quantitative polymerase chain reaction, in 144 fresh tissues including 44 normal and 100 gastric cancer samples. Results: A dramatic overexpression of miR-146a was observed in primary gastric tumors. miR-146a showed lower expression in progressed tumors with greater stages and lymph node metastasis. Conclusion: miR-146a is highly expressed in primary gastric tumor independent of H. pylori infection. It is highly expressed in the lower stages and lymph node-negative tumors. It might suggest the importance of upregulation and downregulation of this miRNA in the initiating/promoting and progressive steps of gastric tumorigenesis, respectively.

Background and purpose One of the most prevalent cancers in the world and Iran is gastric cancer with a high degree of lethality. There are many environmental factors and genes responsible for gastric cancer incidence. Among genetics factors, NDRG1 has been reported to be associated with the gastric cancer progression, while its single nucleotide polymorphisms (SNPs) have not been widely studied. Among the SNPs, rs1049694 might be of interest as it is located in the 3′UTR of NDRG1 transcripts. However, the relevance of rs1049694 with the incidence of gastric cancer has not been studied yet. Materials and methods In this study, 144 patients with gastric cancer and 66 healthy individuals were selected. After taking blood samples and extraction of genomic DNA, genotyping was performed on 1049694 using allele specific primer (ASP)-PCR method. The data were analyzed with the SNPStats and SPSS. Results The results showed that 3 different genotypes were found in gastric cancer rs1049694 NDRG1 gene polymorphism. The rs1049694 allele G was significantly associated with the higher risk of gastric cancer and metastasis. Conclusion The presence of allele G at the rs1049694 position located in NDRG1 gene is significantly associated with the higher risk of gastric cancer. This allele was significantly observed higher in the metastatic patients. This SNP might be studied further to be considered as a biomarker for diagnosis/prognosis of gastric cancer.

Objective Multiple sclerosis (MS) is a chronic disorder involving both inflammatory and neurodegenerative responses. Long non-coding RNAs (lncRNAs) have been had an emerging role as the biomarkers of different disorders, including autoimmune diseases. Previous studies have shown that NR_003531.3 (MEG3a), AC000061.1_201, and AC007182.6 play a role in the pathogenesis of human autoimmune diseases. However, the potential significance of these lncRNAs, as the diagnostic biomarkers of MS, has not been studied yet. We aimed to quantitatively evaluate the expression levels of NR_003531.3, AC000061.1_201, and AC007182.6 in peripheral blood samples of MS patients in comparison with healthy controls. Materials And Methods In this case-control study, the blood samples from 20 MS patients and 10 healthy controls were collected. Total RNA was extracted, and the expression levels of three selected lncRNAs were quantitatively measured using the quantitative real time-polymerase chain reaction (qRT-PCR) method. Results We detected a significant down-regulation in the expression of NR_003531.3 in MS patients, while no marked changes were observed in the expression of AC000061.1_201 and AC007182.6 in patients compared with controls. Based on the receiver operating characteristic (ROC) curve analysis, NR_003531.3 could discriminate MS patients from healthy subjects effectively. Regarding the prognosis of MS patients, NR_003531.3 is significantly and inversely correlated with the expanded disability status scale (EDSS). Conclusion The potential role of NR_003531.3 lncRNA as a diagnostic biomarker to distinguish MS patients is proposed. Prognostically, NR_003531.3 correlates with lower disability rates in MS patients.

Breast cancer as the second most common cancer worldwide tend to be experienced by Iranian women 10 years earlier with a peak incidence at the premenopausal stage. Genetic mutations of TP53 tumor suppressor gene has been shown to be related to early onset of breast cancer. It has been shown already that rs1625895 polymorphic site is related to glioma as well as lung cancer. In this study, we have investigated the role of rs1625895 polymorphism in breast cancer incidence in Iranian women. DNA extraction of 86 breast cancer patients and 96 control individuals have been used for allele-specific primer-PCR and genotyping of allele A and allele G of the TP53 rs1625895. Genotypes frequencies have been shown that GG homozygosis as the most frequent genotype is a significant association with increased risk of breast cancer development in Iranian women (odds ratio = 6, p = 0.002). On the other hand and in comparison to allele G, allele A could cause early death of breast cancer patients by threefolds significantly (p = 0.011). As a conclusion, we show that allele A is the minor allele in both breast cancer patients and also control individuals and major allele G, is related to the increased risk of breast cancer development in Iranian women.

Objective Our study aimed to evaluate the possible association between hsa-mir-499a (rs3746444 A>G) polymorphism and susceptibility to breast cancer in an Isfahanian population. Materials & methods In this case–control study we enrolled 91 healthy subjects and patients with breast cancer. Allele-specific primer PCR was applied for genotyping the SNP. Results Our study showed that the hsa-mir-499a rs3746444 G allele increased the risk of breast cancer regarding to allele frequency differences (OR: 1.922; 95% CI: 1.064–3.470; p = 0.02952) and Armitage's trend test (OR: 1.722; p = 0.04732) in comparison with the A allele. In addition, an in silico attempt to find functional consequences of A>G substation suggested that the G allele may decrease hsa-mir-499a stability based on calculated free energy differences between A and G alleles. Conclusion Our findings illustrated that the mir-499a rs3746444 G polymorphism is associated with higher risk of developing breast cancer in Isfahanian population.

Breast cancer is a major cause of cancer-related death in women worldwide. miRNAs are new players of breast tumorigenesis, used as diagnostic and prognostic biomarkers. Among various miRNAs, miR-126 has been proposed to have a tumor suppressive role in HER2 positive cancer. However, to have a better understanding of its role, further validation is required. The aim of this study was evaluating miR-126 expression level in breast cancer tissues and investigating its potential association with HER2, estrogen and progesterone receptors. miR-126 expression level was measured in 108 specimens including 78 malignant and 30 normal samples using RT-qPCR. The outcome was statistically analyzed. In silico studies were performed to find the potential mechanism of action, through which miR-126 imposes its function. Down-regulation of miR-126 was observed in tumor samples, as compared to the matched normal tissues. Down-regulation of miR-126 was also associated significantly with the absence of estrogen receptor in malignant samples. No association between miR-126 expression and HER2 status was observed. Our in silico analyses showed the possible role of Crk, PI(3)K and Ras proto-oncogenes in breast cancer tumorigenesis. miR-126 is significantly down-regulated in breast cancer tissues. Statistically, it showed no correlation with HER2 positivity. However, the association between lower miR-126 and estrogen receptor negativity was observed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12291-018-0766-6) contains supplementary material, which is available to authorized users.

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.

Despite of promising improvements in treatment of gastric cancer, the mortality rate of this malignancy remains high. Chronic infection by Helicobacter pylori, interfering with intracellular signalling pathways, is the main risk factor for gastric cancer. Some evidence suggests that microRNAs (miRNA), the small noncoding RNA molecules, can play role as oncogenes or tumour suppressors in the cells. MiR-222 is one of the remarkable miRNAs undergoing upregulation in gastric cancer. However, the association between miR-222 upregulation and H. pylori infection in gastric cancer tissues remains unclear. The aim of this study was to analyse the expression level of miR-222 in gastric cancer tissues, evaluating the relationship between miR-222 expression level and H. pylori infection and also finding novel miR-222 targets based on in silico investigations. MiR-222 expression level in 200 patients including 112 H. pylori positive and 88 H. pylori negative was relatively measured using RT-qPCR and compared with 88 healthy samples. In silico enrichment analysis of miR-222 targets was performed by DAVID database to evaluate the possible role(s) of miR-222 in gastric tumourigenesis. We observed upregulated level of miR-222 in gastric cancer tissues compared with normal samples (P

Background: Breast cancer (BC) is the most common neoplasia among females worldwide. Single nucleotide polymorphisms (SNPs) located at the 3′ untranslated Region (3′UTR) can alter gene expression pattern through increasing/decreasing microRNAs (miRNAs) binding energy. Human epidermal growth factor receptor 4 (HER4) can act as either a tumor suppressor or an oncogene in breast cancer. Objectives: We proposed that rs1595065 3′UTR variant of HER4 with a different target binding site of miRNAs may have a correlation with risk of BC phenotypes. In the current study, we aimed to evaluate the association between HER4 rs1595065 3'UTR variant and BC pathological features among the Isfahanian population. Moreover, an in-silico prediction was performed to estimate possible function of the rs1595065. Methods: Overall, 156 patients and controls were genotyped using RFLP-PCR. Armitage test for trend was utilized to investigate the association between rs1595065 and susceptibility to BC. The possible change in the interaction between rs1595065 and microRNAs was studied bioinformatically. Results: Bioinformatics analysis using online tools suggest rs1595065 as a polymorphism in the seed region of four miRNAs binding sites including miR-199a-3p, miR-199b-3p, miR-1244 and miR-3129, and C allele can reduce miRNA-mRNA binding occurrence that may increase HER4 expression. Armitage’s trend test showed that C allele of rs1595065 was significantly associated with HER2 positivity among patients (C allele vs. T allele, OR = 3.111, P = 0.046). Conclusions: rs1595065 could be recommended as a risk factor in regulating HER4 expression and affecting HER2 positivity incidence among BC patients.

Free to read on publisher website ErbB4 can act as either a tumor-suppressor gene or an oncogene in breast cancer. Multiple genetic factors including single nucleotide polymorphisms (SNPs) affect gene expression patterns. Multiple 3'-untranslated region (3'-UTR) SNPs reside within the target binding site of microRNAs, which can strengthen or weaken binding to target genes. The present study aimed to predict potential 3'‑UTR variants of ErbB4 that alter the target binding site of microRNAs (miRNAs) and to clarify the association of the potential variant with the risk of developing breast cancer. In silico prediction was performed to identify potential functional SNPs within miRNA target binding sites in the 3'‑UTR of ErbB4. Thus, 146 patients and controls were genotyped using restriction fragment length polymorphism-polymerase chain reaction. In addition to the Cochran-Armitage test for trend, allele and genotype frequency differences were determined to investigate the association between rs1836724 and the susceptibility to breast cancer. Bioinformatics analysis identified rs1836724 to be a polymorphism in the seed region of four miRNA binding sites (hsa-miR335-5p, hsa-miR-28-5p, has‑miR‑708‑5p and has‑miR‑665), which may participate in the development of breast cancer. Logistic regression data indicated that the T allele of the polymorphism [OR (95% CI)=1.72 (1.056‑2.808), P=0.029] is associated with the risk of breast cancer. Using bioinformatics tools, a correlation was indicated between the presence of the T allele and a reduction in ErbB4 RNA silencing based on miRNA interaction. Furthermore, case subgroup data analysis revealed an association between the C/T genotype and an ER positive phenotype [OR (95% CI)=6.00 (1.082‑33.274), P=0.028] compared with the T/T genotype. ErbB4 and estrogen receptor 1 (ESR1) are regulated by identical miRNAs thus there may be a competition for binding sites. Due to this pattern, if the interaction between miRNAs with one gene is reduced, it may be consistent with the increase in interaction with another one. Therefore, more interaction with rs1836724 C variant within ErbB4 may be associated with higher expression of ESR1 (ER‑positive phenotype). miRNAs interact with ErbB4 mRNA more frequently when it carries C allele at the rs1836724 position compared with the T carriers. Therefore, the identical miRNA interacts with ESR1 less frequently when ErbB4 mRNA has a C allele. Therefore, ESR1 expression may be higher when ErbB4 mRNA has a C allele.