MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

Kulkarni, Varun; Naqvi, A. R.; Uttamani, Juhi Raju; Nares, Salvador

doi:10.3390/ijms17010072

Cited by 23 publications

(22 citation statements)

References 34 publications

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“…On the one hand, this might be related to the cellular model systems. The effects of miRNAs on their target mRNAs strongly depend on the cell type analyzed (52). Thus, it is not surprising that a functional screen of the effects of miRNAs on phagocytosis in PMA-activated THP-1 cells yields different miRNAs compared to transcriptional profiling studies in human monocyte-derived MΦ or murine BMDM populations differentiated ex vivo (11, 12, 15, 37).…”

Section: Discussionmentioning

confidence: 99%

mir-124-5p Regulates Phagocytosis of Human Macrophages by Targeting the Actin Cytoskeleton via the ARP2/3 Complex

et al. 2019

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Phagocytosis is a cellular process crucial for recognition and removal of apoptotic cells and foreign particles, subsequently initiating appropriate immune responses. The process of phagocytosis is highly complex and involves major rearrangements of the cytoskeleton. Due to its complexity and importance for tissue homoeostasis and immune responses, it is tightly regulated. Over the last decade, microRNAs (miRNAs) have emerged as important regulators of biological pathways including the immune response by fine-tuning expression of gene regulatory networks. In order to identify miRNAs implicated in the regulation of phagocytosis, a systematic screening of all currently known, human miRNAs was performed using THP-1 macrophage-like cells and serum-opsonized latex beads. Of the total of 2,566 miRNAs analyzed, several led to significant changes in phagocytosis. Among these, we validated miR-124-5p as a novel regulator of phagocytosis. Transfection with miR-124-5p mimics reduced the number of phagocytic cells as well as the phagocytic activity of phorbol-12-myristate-13-acetate (PMA)-activated THP-1 cells and ex vivo differentiated primary human macrophages. In silico analysis suggested that miR-124-5p targets genes involved in regulation of the actin cytoskeleton. Transcriptional analyses revealed that expression of genes encoding for several subunits of the ARP2/3 complex, a crucial regulator of actin polymerization, is reduced upon transfection of cells with miR-124-5p. Further in silico analyses identified potential binding motifs for miR-124-5p in the mRNAs of these genes. Luciferase reporter assays using these binding motifs indicate that at least two of the genes (ARPC3 and ARPC4) are direct targets of miR-124-5p. Moreover, ARPC3 and ARPC4 protein levels were significantly reduced following miR-124-5p transfection. Collectively, the presented results suggest that miR-124-5p regulates phagocytosis in human macrophages by directly targeting expression of components of the ARP2/3 complex.

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Section: Discussionmentioning

confidence: 99%

mir-124-5p Regulates Phagocytosis of Human Macrophages by Targeting the Actin Cytoskeleton via the ARP2/3 Complex

et al. 2019

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“…As reliable therapeutic targeting requires comprehensive and bona fide target identification, we utilized an innovative, large‐scale and unbiased method to screen for novel miRNAs interacting with components of the hemostatic system. Expression of miRNAs is known to vary greatly between different cell types; in addition, other variables can affect miRNA binding in a cell type‐dependent manner . Thus bona fide identification of miRNAs may critically depend on the experimental system used.…”

Section: Discussionmentioning

confidence: 99%

Large‐scale identification of functional microRNA targeting reveals cooperative regulation of the hemostatic system

Nourse

Braun

Lackner

et al. 2018

Journal of Thrombosis and Haemostasis

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Essentials MicroRNAs (miRNAs) regulate the molecular networks controlling biological functions such as hemostasis. We utilized novel methods to analyze miRNA-mediated regulation of the hemostatic system. 52 specific miRNA interactions with 11 key hemostatic associated genes were identified. Functionality and drugability of miRNA-19b-3p against antithrombin were demonstrated in vivo. SUMMARY: Background microRNAs (miRNAs) confer robustness to complex molecular networks regulating biological functions. However, despite the involvement of miRNAs in almost all biological processes, and the importance of the hemostatic system for a multitude of actions in and beyond blood coagulation, the role of miRNAs in hemostasis is poorly defined. Objectives Here we comprehensively illuminate miRNA-mediated regulation of the hemostatic system in an unbiased manner. Methods In contrast to widely applied association studies, we used an integrative screening approach that combines functional aspects of miRNA silencing with biophysical miRNA interaction based on RNA pull-downs (miTRAP) coupled to next-generation sequencing. Results Examination of a panel of 27 hemostasis-associated gene 3'UTRs revealed the majority to possess substantial Dicer-dependent silencing capability, suggesting functional miRNA targeting. miTRAP revealed 150 specific miRNA interactions with 14 3'UTRs, of which 52, involving 40 miRNAs, were functionally confirmed. This includes cooperative miRNA regulation of key hemostatic genes comprising procoagulant (F7, F8, F11, FGA, FGG and KLKB1) and anticoagulant (SERPINA10, PROZ, SERPIND1 and SERPINC1) as well as fibrinolytic (PLG) components. Bioinformatic analysis of miRNA functionality reveals established and potential novel links between the hemostatic system and other pathologies, such as cancer, bone metabolism and renal function. Conclusions Our findings provide, along with an in-vivo proof of concept, deep insights into the network of miRNAs regulating the hemostatic system and present a foundation for biomarker discovery and novel targeted therapeutics for correction of de-regulated hemostasis and associated processes in the future. A repository of the miRNA targetome covering 14 hemostatic components is provided.

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“…Both cell lines are a suitable cellular model as they are easy to culture and reach very high transfection efficiency. The protein machinery that microRNAs use to regulate gene expression is ubiquitous and we and others have previously shown that this machinery is functional across different cell types37, 39–41. All the experiments were carried out using cells grown in DMEM (Invitrogen) media supplemented with 10% Fetal Calf Serum (Sigma) and 2mM Penicillin/Streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders

Devanna

Chen

et al. 2017

Mol Psychiatry

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Understanding the genetic factors underlying neurodevelopmental and neuropsychiatric disorders is a major challenge given their prevalence and potential severity for quality of life. While large scale genomic screens have made major advances in this area, for many disorders the genetic underpinnings are complex and poorly understood. To date the field has focused predominantly on protein coding variation, but given the importance of tightly controlled gene expression for normal brain development and disorder, variation that affects non-coding regulatory regions of the genome are likely to play an important role in these phenotypes. Herein we show the importance of 3’UTR non-coding regulatory variants across neurodevelopmental and neuropsychiatric disorders. We devised a pipeline for identifying and functionally validating putatively pathogenic variants from NGS data. We applied this pipeline to a cohort of children with severe specific language impairment (SLI) and identified a functional, SLI-associated variant affecting gene regulation in cells and post-mortem human brain. This variant, and the affected gene (ARHGEF39), represent new putative risk factors for SLI. Furthermore, we identified 3’UTR regulatory variants across autism, schizophrenia and bipolar disorder NGS cohorts demonstrating their impact on neurodevelopmental and neuropsychiatric disorders. Our findings show the importance of investigating non-coding regulatory variants when determining risk factors contributing to neurodevelopmental and neuropsychiatric disorders. In the future, integration of such regulatory variation with protein coding changes will be essential for uncovering the genetic causes of complex neurological disorders and the fundamental mechanisms underlying health and disease.

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MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

Cited by 23 publications

References 34 publications

mir-124-5p Regulates Phagocytosis of Human Macrophages by Targeting the Actin Cytoskeleton via the ARP2/3 Complex

mir-124-5p Regulates Phagocytosis of Human Macrophages by Targeting the Actin Cytoskeleton via the ARP2/3 Complex

Large‐scale identification of functional microRNA targeting reveals cooperative regulation of the hemostatic system

Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders

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