Introduction: As the main regulators of gene expression, microRNAs (miRNAs) and transcription factors (TFs) play key roles in complex multifactorial diseases like Multiple Sclerosis (MS). Recently, miRNA/TF-based feed-forward loops (FFLs) have been identified as key elements of biological network motifs in MS. Objectives: Starting from preliminary results obtained by a High-Throughput Next-Generation Sequencing (HT-NGS) approach on an independent MS patients cohort, we aimed to search for miRNA-TF co-regulatory networks involved as hub genetic elements in MS pathogenesis. Methods: The study was performed on peripheral blood samples belonging to 58 MS patients and 20 healthy controls (HCs).To validate the differential expressed (DE) miRNAs resulted from HT-NGS analysis, total RNA extraction was followed by microfluidic qPCR. The subsequent analysis included: miRNA-target gene interaction evaluation using experimentally validated miRNA-target interaction databases (DIANA-Tarbase and miRTarBase) and the output of 5 algorithms (miRanda, DIANA-microT-CDS, RNA22, miRDB and TargetScan) for target genes prediction; miRNA-target experimentally validation using dual luciferase reporter assay; TF-miRNA co-regulatory network identification using miRNA-TF interaction tools (Harmonizome and TransmiR) and a gene-TF interaction database (TRRUST); pathway enrichment analysis using DAVID tool. Results: Microfluidic qPCR analysis revealed 6 significantly DE miRNAs (miR-320a, miR-125a-5p, miR-652-3p, miR-185-5p, miR-942-5p and miR-25-3p) in MS patients compared to HCs. The integrated analysis of miRNAs and computationally predicted mRNAs uncovered 638 miRNA-target pairs validated by reporter gene assays and/or predicted by at least 4 of the 5 interaction tools. Luciferase reporter assays indicated that miR-125a-5p was able to target and modulate DIP2A, ADD2 and E2F2 expression, thus confirming that the in silico predicted target were effectively subjected to miRNA-mediated post-transcriptional modulation. In addition, 409 TF-miRNA interactions were identified, including 198 miRNA-TF FFLs. Furthermore, the enrichment analysis categorized in functional pathways revealed significant representations of networks mostly related to neurotrophin signalling pathway, ErbB signalling pathway and axon guidance. Conclusions: These results uncovered several hub regulatory elements allowing to shed further lights in the gene regulation mechanisms that may underlie the pathogenesis of MS.
MicroRNA and transcription factor co-regulatory network analysis: a combined bioinformatics and molecular strategy for uncovering genetic hub regulatory elements in multiple sclerosis pathogenesis
Nuzziello N;Vilardo L;Pelucchi P;Consiglio A;Liguori M
2018
Abstract
Introduction: As the main regulators of gene expression, microRNAs (miRNAs) and transcription factors (TFs) play key roles in complex multifactorial diseases like Multiple Sclerosis (MS). Recently, miRNA/TF-based feed-forward loops (FFLs) have been identified as key elements of biological network motifs in MS. Objectives: Starting from preliminary results obtained by a High-Throughput Next-Generation Sequencing (HT-NGS) approach on an independent MS patients cohort, we aimed to search for miRNA-TF co-regulatory networks involved as hub genetic elements in MS pathogenesis. Methods: The study was performed on peripheral blood samples belonging to 58 MS patients and 20 healthy controls (HCs).To validate the differential expressed (DE) miRNAs resulted from HT-NGS analysis, total RNA extraction was followed by microfluidic qPCR. The subsequent analysis included: miRNA-target gene interaction evaluation using experimentally validated miRNA-target interaction databases (DIANA-Tarbase and miRTarBase) and the output of 5 algorithms (miRanda, DIANA-microT-CDS, RNA22, miRDB and TargetScan) for target genes prediction; miRNA-target experimentally validation using dual luciferase reporter assay; TF-miRNA co-regulatory network identification using miRNA-TF interaction tools (Harmonizome and TransmiR) and a gene-TF interaction database (TRRUST); pathway enrichment analysis using DAVID tool. Results: Microfluidic qPCR analysis revealed 6 significantly DE miRNAs (miR-320a, miR-125a-5p, miR-652-3p, miR-185-5p, miR-942-5p and miR-25-3p) in MS patients compared to HCs. The integrated analysis of miRNAs and computationally predicted mRNAs uncovered 638 miRNA-target pairs validated by reporter gene assays and/or predicted by at least 4 of the 5 interaction tools. Luciferase reporter assays indicated that miR-125a-5p was able to target and modulate DIP2A, ADD2 and E2F2 expression, thus confirming that the in silico predicted target were effectively subjected to miRNA-mediated post-transcriptional modulation. In addition, 409 TF-miRNA interactions were identified, including 198 miRNA-TF FFLs. Furthermore, the enrichment analysis categorized in functional pathways revealed significant representations of networks mostly related to neurotrophin signalling pathway, ErbB signalling pathway and axon guidance. Conclusions: These results uncovered several hub regulatory elements allowing to shed further lights in the gene regulation mechanisms that may underlie the pathogenesis of MS.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
