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.

Pan-Cancer Analysis of Ligand-Receptor Cross-talk in the Tumor Microenvironment.

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is a key to tumor progression. Here, we deconvoluted bulk tumor transcriptomes to infer cross-talk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in situ hybridization and IHC data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune cross-talk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of cross-talk in the TME. SIGNIFICANCE: This study provides deconvoluted bulk tumor transcriptomes across multiple cancer types to infer cross-talk in the tumor microenvironment.

Systematic bioinformatic approaches reveal novel gene expression signatures associated with acquired resistance to EGFR targeted therapy in lung cancer.

OBJECTIVES: Human non-small cell lung cancer (NSCLC) that harbors activating mutations in epidermal growth factor receptor (EGFR) initially responds to treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib but eventually tumor cells acquire resistance. To date, several gene expression profiles have been reported in TKIs-resistant EGFR-mutant NSCLC. The objective of this study is to identify robust gene expression signatures, biological processes, and promising overcoming targets for TKIs-resistant EGFR-mutant NSCLC. MATERIALS AND METHODS: Five publicly available microarray datasets were integrated by performing two network-based meta-analyses following by protein-protein interaction (PPI) network and gene set enrichment analysis. RESULTS AND CONCLUSION: According to our meta-analyses, 830 and 1286 genes were differentially expressed in the TKIs-resistant EGFR-mutant NSCLC cell lines compared to TKIs-sensitive EGFR-mutant NSCLC cell lines in the absence and presence of TKIs treatment, respectively. PPI network analysis identified ESR1 and ELAVL1 to be the most highly ranked hub genes involved in the NSCLC acquired TKI-resistance. Moreover, gene set enrichment analyses indicated that up-regulated genes are mainly distributed in hallmarks "Glycolysis", some "E2F targets". Down-regulated genes mainly contribute to hallmarks "interferon alpha response", "interferon gamma response", and also "E2F targets". For the first time, this study has demonstrated several robust candidate genes and pathways of the NSCLC acquired TKI-resistance. Further experimental verifications are highly recommended to examine 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.

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.

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.

FISH Analysis for del6q21 and del17p13 in B-cell Chronic Lymphocytic Leukemia in Iranians.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. Major progress has been made in assessing typical chromosomal abnormalities and recognition of the correlation of these chromosomal abnormalities with laboratory features and clinical course of the disease. The most frequent genomic changes are deletions at 13q14, 11q22-23 and 17p13 and trisomy of chromosome 12. OBJECTIVES: The aim of this study was to investigate the frequency of chromosomal aberrations in B-CLL patients' peripheral blood and/or bone marrow using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH) and to evaluate the correlation between these genomic changes and clinical findings. PATIENTS AND METHODS: I-FISH analyses were performed on bone marrow and blood samples of 66 B-CLL patients. RESULTS: Deletion of 17p13 was found in 11 (16.6%) and deletion 6q21 was present in 5 (7.5%). Statistical analyses were performed to investigate the correlation of these molecular-cytogenetic findings with family history, Rai staging and CD38 marker. No clear differences in distribution was noted for del17p13 and del6q21 among patients with and without family history, and no direct correlation was noted between these genomic changes and CD38 marker, but the correlation of del17p13 and Rai stage was significant. There was a high frequency of Rai stage II within del17p13 patients. CONCLUSIONS: It was demonstrated that the presence of del6q21 in B-CLL patients indicates poor prognosis and on the contrary, presence of del17p13 points at the good prognostic value of the disease.