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MiR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway.

ABSTRACT: Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated ?IIb?3 activation and ?-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of ?IIb?3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced ?IIb?3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcR?-mediated phosphorylation of Syk and PLC?2, GPVI downstream signaling molecules, but effects of miR-15a-5p on ?IIb?3 activation did not extend to other ITAM-signaling receptors (Fc?RIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential "master-miR" for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.

SUBMITTER: Basak I

PROVIDER: S-EPMC6397079 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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miR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway.

Basak Indranil I Bhatlekar Seema S Manne Bhanu K BK Stoller Micelle M Hugo Sarah S Kong X X Ma L L Rondina Matthew T MT Weyrich Andrew S AS Edelstein Leonard C LC Bray Paul F PF

Journal of thrombosis and haemostasis : JTH 20190225 3

Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated αIIbβ3 activation and α-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhi ...[more]

PMID: 30632265

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Project description:Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high-risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR-15a-5p, miR-15b-5p, and miR-16-5p (hereafter miR-15a, miR-15b or miR-16) were down-regulated in patient-derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR-15. Moreover, overexpression of miR-15a, miR-15b or miR-16 significantly reduced the levels of MYCN mRNA and N-Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N-Myc protein levels, as well as increasing mRNA half-life in NB cells. By performing immunoprecipitation assays of argonaute-2 (Ago2), a core component of the RNA-induced silencing complex, we showed that miR-15a, miR-15b and miR-16 interact with MYCN mRNA. Luciferase reporter assays showed that miR-15a, miR-15b and miR-16 bind with 3'UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR-15a, miR-15b and miR-16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR-15a-, miR-15b- and miR-16-expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR-15a, miR-15b and miR-16 exert a tumor-suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

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MiR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway.

Publications

miR-15a-5p regulates expression of multiple proteins in the megakaryocyte GPVI signaling pathway.

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