ErbB4 3'-UTR Variant (c.*3622A>G) is Associated with ER/PR Negativity and Advanced Breast Cancer.

A genetic variant may alter a gene expression level and as a result be associated with pathological characteristics in breast cancer. In this research, the frequency and association of the ErbB4 3'-untranslated region (3'-UTR) variant, rs12471583 (c.*3622A>G) was studied in an Iranian breast cancer patients. In silico assessment was performed to predict the function of the rs12471583 variant located on the 3'-UTR of ErbB4. Furthermore, as a case-control study, this polymorphism was genotyped in 243 breast cancer patients and non-cancerous controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The Armitage's trend test and regular association tests were performed to analyze a possible association between the rs12471583 and risk of breast cancer and its relevant pathological traits. The bioinformatics analysis predicted that the rs12471583 SNP is located on the four miRNA binding sites, including miR-511-5p, miR-4659a-5p, miR-4659b-5p, and miR-6830-3p. According to logistic regression tests, the G allele is negatively associated with ER- (OR = 0.20, 95% C.I. = 0.04-0.93, p = 0.026), PR- (OR = 0.31, 95% C.I. = 0.10-0.98, p = 0.039), ER-/PR- (OR = 0.20, 95% C.I. = 0.04-0.93, p = 0.026), and advanced breast cancer (OR = 0.40, 95% C.I. = 0.18-0.85, p = 0.016). It has been found that ErbB4 expression may be linked to unfavorable outcomes in breast cancer. Likewise, our results suggest that the G allele may strengthen miRNA-ErbB4 binding efficiency and as a result reduce expression of ErbB4. This is a possible explanation for the observed association.

Integrative meta-analysis of publically available microarray datasets of several epithelial cell lines identifies biological processes affected by silver nanoparticles exposure.

The present study aimed to identify differentially expressed genes (DEGs) under silver nanoparticle (AgNPs) treatment. We used a meta-analysis approach to integrate four publicly available microarray datasets, containing control and epithelium samples treated by either AgNPs- or Ag ions. The Fisher's method combined p-values of studies. Post hoc analyses including protein-protein interaction (PPI) and the overrepresentation test were conducted. Analytical results identified 1652 DEGs associated with AgNPs exposure. The most significant up-regulated genes, including MT1H, MT1X, and MT2A were metallothionein family members. The most significant down-regulated gene, TM4SF5, is a novel biomarker for AgNPs exposure. The PPI network analysis revealed that a member of the heat shock protein family, HSP90AA1, is the top up-regulated "hub" gene. Up-regulation of heat shock proteins and metallothionein genes is part of a cellular response to oxidative stress induced by AgNPs treatment. Interestingly, AgNPs may interact negatively with blood coagulation and amino acid metabolism systems.

Association of a potential functional mir-520f rs75598818 G>A polymorphism with breast cancer.

Some of the single-nucleotide polymorphisms in miRNA genes have been studied to date to find their association with the risk of breast cancer (BC). However, no study has been conducted to investigate the association of the mir-520f rs75598818G>A in BC. In the present study, rs75598818 association with BC in an Iranian population has been investigated, and an in silico analysis was performed to predict the function of rs75598818 polymorphism in BC. The rs75598818 was genotyped in 129 BC patients and 144 healthy women, using the PCR-RFLP method. The frequency of alleles and genotypes were considered to find the associations between rs75598818 alleles/genotypes, and BC risk and pathological characteristics of the patients. Statistical analysis showed that the rs75598818 GA genotype was significantly associated with BC (GA versus GG, OR=0.50, 95% CI: 0.25-0.98, P =0.041), highstage BC (stage III/IV versus I/II, GA versus GG, OR=0.27, 95% CI: 0.09-0.81, P =0.015), and HER-2 positive status (GA versus GG, OR=19.00, 95% CI: 4.64-77.82, P <0.001). Notably, the rs75598818 GA genotype has a negative association pattern since it reduces the risk of BC and high stage BC. Conversely, it increases the risk of HER-2 positivity. Computational results suggested that the rs75598818 polymorphism affects the stability of mir-520f stem-loop and as a result miR-520f-3p production that is a potential tumour suppressor. A contribution of the mir-520f rs75598818 polymorphism to BC had been unexplored before. In the present study, we performed an association study and a bioinformatics approach to evaluate this polymorphism in BC. However, further functional experiments and large-scale association studies with various ethnicities are required to elaborate our findings.

ABCC4 functional SNP in the 3' splice acceptor site of exon 8 (G912T) is associated with unfavorable clinical outcome in children with acute lymphoblastic leukemia.

OBJECTIVES: ATP-binding cassette subfamily C member 4 (ABCC4) encoding MRP4 protein is involved in pediatric acute lymphoblastic leukemia (ALL) drug resistance. The nonsynonymous single nucleotide polymorphism (SNP) rs2274407 (G912T; K304N) is located in the 3' splice acceptor site of exon 8 of ABCC4 pre-mRNA. The aim of this study was to investigate the prognostic value of rs2274407 in childhood ALL and its possible functional effect on MRP4. METHODS: ABCC4 G912T SNP was genotyped in 145 Iranian Philadelphia-negative (Ph-) children with ALL using modified tetra-primer ARMS PCR and evaluated for possible association with 3-year disease-free survival (3DFS). In addition, functional impact of rs2274407 on the MRP4 activity and possible post-transcriptional modifications were bioinformatically and experimentally studied. RESULTS: ABCC4 912T allele carriers (G/T and T/T genotypes) are associated with worse 3DFS in Pre-B cell ALL [P = 0.00019, OR (95% CI) = 13.17 (2.55-68.11)]. In addition, computational studies showed that K304N alteration has no impact on the MRP4 activity. However, it may disrupt the normal splicing process of ABCC4 pre-mRNA. CONCLUSIONS: To date, this is the first study that shows the potential functional impact of rs2274407 SNP on the aberrant splicing of ABCC4 mRNA. We also demonstrated a robust association between G912T and pediatric ALL negative outcome, which may be explained by the novel computational studies performed in this study.

Aberrant expression of miR-9 in benign and malignant breast tumors.

PURPOSE: MicroRNAs (miRNAs) are involved in the progression of breast cancer (BC). miR-9 has been reported to be correlated with either favorable or unfavorable events in BC. This study was aimed to evaluate the expression level of miR-9 in human breast tissues, including benign and malignant tumor samples and also healthy tissue. MATERIALS AND METHODS: The expression level of miR-9 was analyzed in 10 normal breast tissues, 30 malignant, and 30 benign breast tumor tissue samples using RT-PCR and qPCR. In addition, bioinformatics assessment upon miR-9 functionality in BC cells was performed. RESULTS AND DISCUSSION: The miR-9 expression level was downregulated in tumor tissues, including benign and malignant compared to the healthy tissue was observed (P value, < 0.0001; fold change, -1.37). In addition, miR-9 expression level was reduced in benign tumors compared with malignant tumors (P value, < 0.0001; fold change, -1.35). Moreover, according to the AUCs (area under curve) of receiver operating characteristic (ROC) curves, miR-9 showed significant capability for distinguishing benign from healthy, malignant from healthy, benign from malignant, and tumor from health tissues. Furthermore, pathways in cancer, p53 signaling pathway, and focal adhesion were manifested by computational analysis as miR-9 related signaling pathways which have logical association with experimental observations. CONCLUSION: In conclusion, downregulation of miR-9 in benign tumors vs healthy tissue and its overexpression in malignant tumors vs benign tumors suggest paradoxical functionality for this miRNA. Our results shed additional information on controversial expression pattern of miR-9 depending on different progression level of BC.

Evaluation of rs62527607 [GT] single nucleotide polymorphism located in BAALC gene in children with acute leukemia using mismatch PCR-RFLP.

Acute leukemia is the most common cancer in children and involves several factors that contribute to the development of multidrug resistance and treatment failure. According to our recent studies, the BAALC gene is identified to have high mRNA expression levels in childhood acute lymphoblastic leukemia (ALL) and those with multidrug resistance. Several polymorphisms are associated with the expression of this gene. To date, there has been no study on the rs62527607 [GT] single nucleotide polymorphism (SNP) of BAALC gene and its link with childhood acute lymphoblastic and myeloid leukemia (AML). The purpose of this study is to evaluate the prevalence of this polymorphism in pediatric acute leukemia, as well as its relationship with prognosis. DNA samples were extracted from bone marrow slides of 129 children with ALL and 16 children with AML. The rs62527607 [GT] SNP was evaluated using mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based analysis. The association between the SNP alleles and patient disease-free survival was then assessed. The prevalence of the T-allele of rs62527607 [GT] SNP in childhood T-ALL and pre-B-ALL was 28.3% and 11.2%, respectively. In the pre-B-ALL patients, 3 year disease free survival was associated with the GG genotype. Results showed a robust association between the rs62527607 SNP and the risk of relapse in ALL, but not AML, patients. T-ALL patients with the GT genotype had an 8.75 fold higher risk of relapse. The current study demonstrates a significant association between the genotype GT and the polymorphic allele G424T, and introduces this SNP as a negative prognostic factor in children with ALL.

Tumor-promoting function of single nucleotide polymorphism rs1836724 (C3388T) alters multiple potential legitimate microRNA binding sites at the 3'-untranslated region of ErbB4 in breast cancer.

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, has­miR­708­5p and has­miR­665), 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 (1.056­2.808), 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 (1.082­33.274), 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 (ER­positive 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.

Integrative computational in-depth analysis of dysregulated miRNA-mRNA interactions in drug-resistant pediatric acute lymphoblastic leukemia cells: an attempt to obtain new potential gene-miRNA pathways involved in response to treatment.

Acute lymphoblastic leukemia (ALL) is the major neoplasia type among children. Despite the tremendous success of current treatment strategies, drug resistance still remains a major cause of chemotherapy failure and relapse in pediatric patients. Overwhelming evidence illustrates that microRNAs (miRNAs) act as post-transcriptional regulators of drug-resistance-related genes. The current study was aimed at how dysregulated miRNA-mRNA-signaling pathway interaction networks mediate resistance to four commonly used chemotherapy agents in pediatric ALL, including asparaginase, daunorubicin, prednisolone, and vincristine. Using public expression microarray datasets, a holistic in silico approach was utilized to investigate candidate drug resistance miRNA-mRNA-signaling pathway interaction networks in pediatric ALL. Our systems biology approach nominated significant drug resistance and cross-resistance miRNAs, mRNAs, and cell signaling pathways based on anti-correlative relationship between miRNA and mRNA expression pattern. To sum up, our systemic analysis disclosed either a new potential role of miRNAs, or a possible mechanism of cellular drug resistance, in chemotherapy resistance of pediatric ALL. The current study may shed light on predicting drug response and overcoming drug resistance in childhood ALL for subsequent generations of chemotherapies.

Modified tetra-primer ARMS PCR as a single-nucleotide polymorphism genotyping tool.

OBJECTIVES: Genotyping of single-nucleotide polymorphisms (SNPs) has been applied in various genetic contexts. Tetra-primer amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) is reported as a prominent assay for SNP genotyping. However, there were published data that may question the reliability of this method on some occasions, in addition to a laborious and time-consuming procedure of the optimization step. In the current study, a new SNP genotyping method named modified tetra-primer ARMS (MTPA) PCR was developed based on tetra-primer ARMS PCR. DESIGN AND METHODS: The modified method has two improvements in its instruction, including equalization of outer primer and inner primer strength by additional mismatch in outer primers, and consideration of equal annealing temperature of specific fragments more than melting temperature of primers. Advantageously, a new computer software was provided for designing primers based on novel concepts. RESULTS: The usual tetra-primer ARMS PCR has a laborious process for optimization. In nonoptimal PCR programs, identification of the accurate genotype was found to be very difficult. However, in MTPA PCR, equalization of the amplicons and primer strength leads to increasing specificity and convenience of genotyping, which was validated by sequencing. CONCLUSIONS: In the MTPA PCR technique, a new mismatch at -2 positions of outer primers and equal annealing temperature improve the genotyping procedure. Together, the introduced method could be suggested as a powerful tool for genotyping single-nucleotide mutations and polymorphisms.

Genotyping data and novel haplotype diversity of STR markers in the SLC26A4 gene region in five ethnic groups of the Iranian population.

BACKGROUND AND AIMS: SLC26A4 gene mutations are the second currently identifiable genetic cause of autosomal recessive nonsyndromic hearing loss after GJB2 mutations. Because of the extensive size of the SLC26A4 gene and the variety of mutations, indirect diagnosis using linkage analysis has been suggested. Therefore, in this investigation three potential short tandem repeat (STR) markers related to this region including D7S2420, D7S496, and D7S2459 were selected for further analysis. METHODS: The characteristics and haplotype frequency of the markers were examined for the first time in five ethnic groups of the Iranian population including Fars, Azari, Turkmen, Gilaki, and Arab using the polymerase chain reaction followed by fluorescent capillary electrophoresis. RESULTS were analyzed by GeneMarker HID Human STR Identity, GenePop, Microsatellite tools, PowerMarker 3.25, and Arlequin 3.5 software. RESULTS: Analysis of the allelic frequency revealed the presence of 11, 10, and 8 alleles for D7S2420, D7S496, and D7S2459 markers, respectively, in the Iranian population. The detailed analysis of each ethnic group was reported. Calculated polymorphism information content values were above 0.7 in the Iranian population. Pairwise linkage disequilibrium (LD) revealed a significant LD in pairing markers of D7S2420-D7S496 and in D7S496-D7S2459. Estimation of the haplotype frequency showed the presence of 20, 13, 15, 15, and 20 informative haplotypes in Fars, Azari, Turkmen, Gilaki, and Arabian ethnics, respectively. CONCLUSION: Together, the investigated markers could be suggested as powerful tools for linkage analysis of SLC26A4 gene mutations in the Iranian population.