title: miR-125b modulates megakaryocyte maturation by targeting the cell-cycle inhibitor p19INK4D.
abstract: A better understanding of the mechanisms involved in megakaryocyte maturation will facilitate the generation of platelets in vitro and their clinical applications. A microRNA, miR-125b, has been suggested to have important roles in the self-renewal of megakaryocyte-erythroid progenitors and in platelet generation. However, miR-125b is also critical for hematopoietic stem cell self-renewal. Thus, the function of miR-125b and the complex signaling pathways regulating megakaryopoiesis remain to be elucidated. In this study, an attentive examination of the endogenous expression of miR-125b during megakaryocyte differentiation was performed. Accordingly, the differentiation of hematopoietic stem cells requires the downregulation of miR-125b, whereas megakaryocyte determination and maturation synchronize with miR-125b accumulation. The overexpression of miR-125b improves megakaryocytic differentiation of K562 and UT-7 cells. Furthermore, stage-specific overexpression of miR-125b in primary cells demonstrates that miR-125b mediates an enhancement of megakaryocytic differentiation after megakaryocyte determination, the stage at which megakaryocytes are negative for the expression of the hematopoietic progenitor marker CD34. The identification of miR-125b targets during megakaryopoiesis was focused on negative regulators of cell cycle because the transition of the G1/S phase has been associated with megakaryocyte polyploidization. Real-time PCR, western blot and luciferase reporter assay reveal that p19INK4Dis a direct target of miR-125b. P19INK4Dknockdown using small interfering RNA (siRNA) in megakaryocyte-induced K562 cells, UT-7 cells and CD61+promegakaryocytes results in S-phase progression and increased polyploidy, as well as improved megakaryocyte differentiation, similarly to the effects of miR-125b overexpression. P19INK4Doverexpression reverses these effects, as indicated by reduced expression of megakaryocyte markers, G1-phase arrest and polyploidy decrease. P19INK4Dknockdown in miR-125b downregulated cells or p19INK4Doverexpression in miR-125b upregulated cells rescued the effect of miR-125b. Taken together, these findings suggest that miR-125b expression positively regulates megakaryocyte development since the initial phases of megakaryocyte determination, and p19INK4Dis one of the key mediators of miR-125b activity during the onset of megakaryocyte polyploidization.
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
authors.forename: Mingyi
authors.initials: M
authors.lastname: Qu
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Fang
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|Southern Medical University, Guangzhou 510005, China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Zou
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Zeng
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Zeng
authors.initials: Z
authors.lastname: Fan
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Yue
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xie
authors.affiliation: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing 100850, China.|South China Research Center for Stem Cell and Regenerative Medicine, South China Institute Biomedicine, Guangzhou 510005, China.
authors.forename: Xuetao
authors.initials: X
authors.lastname: Pei
mesh_terms: D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D003114:Colony-Forming Units Assay; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology; D016193:G1 Phase / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011123:Polyploidy; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: C493974:CDKN2D protein, human; D050764:Cyclin-Dependent Kinase Inhibitor p19; C531888:MIRN125 microRNA, human; D035683:MicroRNAs; D013755:Tetradecanoylphorbol Acetate