title: Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells.
abstract: Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the β-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; α2γ2) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the β-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate γ-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the γ-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a γ-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of γ-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total- and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.
authors.affiliation: Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA 30912, USA Department of Pediatrics, Augusta University, Augusta, GA 30912, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Ward
authors.affiliation: Department of Pediatrics, Augusta University, Augusta, GA 30912, USA.
authors.forename: Biaoru
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA 30912, USA Department of Pediatrics, Augusta University, Augusta, GA 30912, USA bpace@gru.edu.
authors.forename: Betty S
authors.initials: BS
authors.lastname: Pace
mesh_terms: D020413:3' Untranslated Regions; D005319:Fetal Hemoglobin / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D020868:Gene Silencing; D006021:Glycophorins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D035683:MicroRNAs / Q000502:physiology*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor / Q000502:physiology; D053833:Wetlands
chemical_list: D020413:3' Untranslated Regions; D006021:Glycophorins; D051741:Kruppel-Like Transcription Factors; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D017467:Receptors, Erythropoietin; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C080774:erythroid Kruppel-like factor; D005319:Fetal Hemoglobin
keywords: STAT3; fetal hemoglobin; gamma-globin; miR-34a