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.
PlattOSBrambillaDJRosseWFMilnerPFCastroOSteinbergMHKlugPP. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med1994; 330: 1639–44.
2.
PoillonWNKimBCRodgersGPNoguchiCTSchechterAN. Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations. Proc Natl Acad Sci U S A1993; 90: 5039–43.
3.
StamatoyannopoulosGMajerusPWPerlmutterRMVarmusH. The molecular basis of blood diseases, 3rd ed. Phildadelphia: W.B. Saunders Company, 2001.
4.
SankaranVGMenneTFXuJAkieTELettreGVan HandelBMikkolaHKHirschhornJNCantorABOrkinSH. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science2008; 322: 1839–42.
5.
ZhouDLiuKSunCWPawlikKMTownesTM. KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching. Nat Genet2010; 42: 742–4.
6.
SattaSPerseuLMoiPAsunisICabrioluAMaccioniLDemartisFRManunzaLCaoAGalanelloR. Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin. Haematologica2011; 96: 767–70.
7.
BorgJPapadopoulosPGeorgitsiMGutierrezLGrechGFanisPPhylactidesMVerkerkAJvan der SpekPJScerriCACassarWGaldiesRvan IjckenWOzgürZGillemansNHouJBugejaMGrosveldFGvon LindernMFeliceAEPatrinosGPPhilipsenS. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet2010; 42: 801–5.
8.
GallienneAEDreauHMSchuhAOldJMHendersonS. Ten novel mutations in the erythroid transcription factor KLF1 gene associated with increased fetal hemoglobin levels in adults. Haematologica2012; 97: 340–3.
9.
Harju-BakerSCostaFCFedosyukHNeadesRPetersonKR. Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site. Mol Cell Biol2008; 28: 3101–13.
10.
TanabeOKatsuokaFCampbellADSongWYamamotoMTanimotoKEngelD. An embryonic/fetal beta-type globin gene repressor contains a nuclear receptor TR2/TR4 heterodimer. Embo J2002; 21: 3434–42.
11.
CuiSKolodziejKEObaraNAmaral-PsarrisADemmersJShiLEngelJDGrosveldFStrouboulisJTanabeO. Nuclear receptors TR2 and TR4 recruit multiple epigenetic transcriptional corepressors that associate specifically with the embryonic beta-type globin promoters in differentiated adult erythroid cells. Mol Cell Biol2011; 31: 3298–311.
12.
WeinbergRSJiXSuttonMPerrineSGalperinYLiQLiQLiebhaberSAStamatoyannopoulosGAtwehGF. Butyrate increases the efficiency of translation of gamma-globin mRNA. Blood2005; 105: 1807–9.
13.
HahnCKLowreyCH. Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA. Blood2014; 124: 2730–4.
14.
AzzouziIMoestHWinklerJFauchereJCGerberAPWollscheidBStoffelMSchmuggeMSpeerO. MicroRNA-96 directly inhibits gamma-globin expression in human erythropoiesis. PLoS One2011; 6: 28–28.
15.
LeeYTde VasconcellosJFYuanJByrnesCNohSJMeierERKimKSRabelAKaushalMMuljoSAMillerJL. LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo. Blood2013; 122: 1034–41.
16.
BetelDKoppalAAgiusPSanderCLeslieC. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol2010; 11: 27–27.
17.
LewisBPBurgeCBBartelDP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell2005; 120: 15–20.
18.
FriedmanRCFarhKKBurgeCBBartelDP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res2009; 19: 92–105.
19.
GarciaDMBaekDShinCBellGWGrimsonABartelDP. Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs. Nat Struct Mol Biol2011; 18: 1139–46.
20.
SangermanJLeeMSYaoXOtengEHsiaoCHLiWZeinSOfori-AcquahSFPaceBS. Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves gamma-globin activation by CREB1 and ATF-2. Blood2006; 108: 3590–9.
21.
LivakKJSchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods2001; 25: 402–8.
22.
GrilloGTuriALicciulliFMignoneFLiuniSBanfiSGennarinoVAHornerDSPavesiGPicardiEPesoleG. UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs. Nucleic Acids Res2010; 38: 30–30.
23.
BrenneckeJStarkARussellRBCohenSM. Principles of microRNA-target recognition. PLoS Biol2005; 3: e85–e85.
24.
ChoongMLYangHHMcNieceI. MicroRNA expression profiling during human cord blood-derived CD34 cell erythropoiesis. Exp Hematol2007; 35: 551–64.
25.
ChenSYWangYTelenMJChiJT. The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS One2008; 3: e2360–e2360.
26.
ZhanMMillerCPPapayannopoulouTStamatoyannopoulosGSongCZ. MicroRNA expression dynamics during murine and human erythroid differentiation. Exp Hematol2007; 35: 1015–25.
27.
IchimuraARuikeYTerasawaKShimizuKTsujimotoG. MicroRNA-34a inhibits cell proliferation by repressing mitogen-activated protein kinase kinase 1 during megakaryocytic differentiation of K562 cells. Mol Pharmacol2010; 77: 1016–24.
28.
KosakaNSugiuraKYamamotoYYoshiokaYMiyazakiHKomatsuNOchiyaTKatoT. Identification of erythropoietin-induced microRNAs in haematopoietic cells during erythroid differentiation. Br J Haematol2008; 142: 293–300.
29.
NavarroFGutmanDMeireECaceresMRigoutsosIBentwichZLiebermanJ. miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53. Blood2009; 114: 2181–92.
30.
SunFFuHLiuQTieYZhuJXingRSunZZhengX. Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett2008; 582: 1564–8.
31.
ChristoffersenNRShalgiRFrankelLBLeucciELeesMKlausenMPilpelYNielsenFCOrenMLundAH. p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell Death Differ2010; 17: 236–45.
32.
WengWWangMXieSLongYLiFSunFYuYLiZ. YY1-C/EBPalpha-miR34a regulatory circuitry is involved in renal cell carcinoma progression. Oncol Rep2014; 31: 1921–7.
33.
ChenQRYuLRTsangPWeiJSSongYKCheukAChungJYHewittSMVeenstraTDKhanJ. Systematic proteome analysis identifies transcription factor YY1 as a direct target of miR-34a. J Proteome Res2011; 10: 479–87.
34.
ZhaoJLammersPTorranceCJBaderAG. TP53-independent function of miR-34a via HDAC1 and p21(CIP1/WAF1). Mol Ther2013; 21: 1678–86.
35.
TivnanATraceyLBuckleyPGAlcockLCDavidoffAMStallingsRL. MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma. BMC Cancer2011; 11: 1471–2407.
36.
LalAThomasMPAltschulerGNavarroFO'DayELiXLConcepcionCHanYCThieryJRajaniDKDeutschAHofmannOVenturaAHideWLiebermanJ. Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet2011; 7: e1002363–e1002363.
37.
LeeYTde VasconcellosJFByrnesCKaushalMRabelATumburuLAllwardtJMMillerJL. Erythroid-Specific Expression of LIN28A is sufficient for robust gamma-globin gene and protein expression in adult erythroblasts. PLoS One2015; 10: e0144977–e0144977.
38.
SankaranVGMenneTFScepanovicDVergilioJAJiPKimJThiruPOrkinSHLanderESLodishHF. MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13. Proc Natl Acad Sci U S A2011; 108: 1519–24.
39.
LulliVRomaniaPMorsilliOCianciulliPGabbianelliMTestaUGiulianiAMarzialiG. MicroRNA-486-3p regulates gamma-globin expression in human erythroid cells by directly modulating BCL11A. PLoS One2013; 8: e60436–e60436.
40.
BianchiNZuccatoCLamprontiIBorgattiMGambariR. Expression of miR-210 during erythroid differentiation and induction of gamma-globin gene expression. BMB Rep2009; 42: 493–9.
41.
WalkerALStewardSHowardTAMortierNSmeltzerMWangYDWareRE. Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia. Blood2011; 118: 5664–70.
42.
SangokoyaCTelenMJChiJT. microRNA miR-144 modulates oxidative stress tolerance and associates with anemia severity in sickle cell disease. Blood2010; 116: 4338–48.
43.
LiBTorresCMTakezakiMNeunertCKutlarAPaceBS. Micro RNA profiling to identify regulator of fetal hemoglobin expression in sickle cell disease. Blood2014; 124: 49–49.
44.
RothschildSI. microRNA therapies in cancer. Mol Cell Ther2014; 2: 2052–8426.
45.
JoplingCLYiMLancasterAMLemonSMSarnowP. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science2005; 309: 1577–81.
46.
RhyasenGWStarczynowskiDT. Deregulation of microRNAs in myelodysplastic syndrome. Leukemia2012; 26: 13–22.
47.
Madrigal-MatuteJRotllanNArandaJFFernandez-HernandoC. MicroRNAs and atherosclerosis. Curr Atheroscler Rep2013; 15: 0322–0322.
48.
ChangTCWentzelEAKentOARamachandranKMullendoreMLeeKHFeldmannGYamakuchiMFerlitoMLowensteinCJArkingDEBeerMAMaitraAMendellJT. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell2007; 26: 745–752.
49.
TarasovVJungPVerdoodtBLodyginDEpanchintsevAMenssenAMeisterGHermekingH. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle2007; 6: 1586–1593.
50.
AgostiniMKnightRA. miR-34: from bench to bedside. Oncotarget2014; 5: 872–81.
51.
BaderAG. miR-34 – a microRNA replacement therapy is headed to the clinic. Front Genet2012; 3: 120–120.
52.
AmbrosV. The functions of animal microRNAs. Nature2004; 431: 350–5.
53.
OromUANielsenFCLundAH. MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell2008; 30: 460–71.
54.
PlaceRFLiLCPookotDNoonanEJDahiyaR. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A2008; 105: 1608–13.
55.
GumucioDLHeilstedt-WilliamsonHGrayTATarleSASheltonDATagleDASlightomJLGoodmanMCollinsFS. Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes. Mol Cell Biol1992; 12: 4919–29.
56.
FengQZhangY. The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes. Genes Dev2001; 15: 827–32.
57.
GnanapragasamMNScarsdaleJNAmayaMLWebbHDDesaiMAWalavalkarNMWangSZZu ZhuSGinderGDWilliamsDCJr. p66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex. Proc Natl Acad Sci USA2011; 108: 7487–92.
58.
PaceBSZeinS. Understanding mechanisms of gamma-globin gene regulation to develop strategies for pharmacological fetal hemoglobin induction. Dev Dyn2006; 235: 1727–37.
YaoXKodeboyinaSLiuLDzanduJSangermanJOfori-AcquahSFPaceBS. Role of STAT3 and GATA-1 interactions in gamma-globin gene expression. Exp Hematol2009; 37: 889–900.
61.
NamTGLeeJWalkerJRBrinkerAChoCYSchultzPG. Identification and characterization of small-molecule inducers of fetal hemoglobin. Chem Med Chem2011; 6: 777–80.
62.
WierengaATVogelzangIEggenBJVellengaE. Erythropoietin-induced serine 727 phosphorylation of STAT3 in erythroid cells is mediated by a MEK-, ERK-, and MSK1-dependent pathway. Exp Hematol2003; 31: 398–405.
