title: Expression of regulatory platelet microRNAs in patients with sickle cell disease.
abstract: BACKGROUND
  Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile.
  
  
  RESULTS
  We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1.
  
  
  CONCLUSIONS
  We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets.
authors.affiliation: Division of Pediatric Hematology-Oncology, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Jain
authors.forename: Maria G
authors.initials: MG
authors.lastname: Kapetanaki
authors.forename: Nalini
authors.initials: N
authors.lastname: Raghavachari
authors.forename: Kimberly
authors.initials: K
authors.lastname: Woodhouse
authors.forename: Guoying
authors.initials: G
authors.lastname: Yu
authors.forename: Suchitra
authors.initials: S
authors.lastname: Barge
authors.forename: Claudia
authors.initials: C
authors.lastname: Coronnello
authors.forename: Panayiotis V
authors.initials: PV
authors.lastname: Benos
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kato
authors.forename: Naftali
authors.initials: N
authors.lastname: Kaminski
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
mesh_terms: D000328:Adult; D000368:Aged; D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000473:pathology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002883:Chromosomes, Human, Pair 14 / Q000378:metabolism; D019295:Computational Biology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D018392:Genomic Imprinting; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008297:Male; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D058977:Molecular Sequence Annotation; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D014262:Tricuspid Valve Insufficiency / Q000235:genetics / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D055815:Young Adult
chemical_list: D035683:MicroRNAs; D006918:Hydroxyurea