title: MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells.
abstract: Human cardiomyocyte progenitor cells (hCMPCs) are cardiac progenitor cells that are unique for their efficient differentiation into beating cardiomyocytes without requiring co-culture with neonatal cardiomyocytes. hCMPCs have shown great potential in preserving the function of infarcted mouse myocardium. MiRNA-204 has been reported to be up-regulated in differentiated hCMPCs, however, its biological significance is unclear. In this study, hCMPC proliferation, viability, apoptosis and necrosis were determined using the ELISA Kit (colorimetric BrdU detection), Cell Counting Kit-8, and Annexin V and propidium iodide staining, respectively. MiRNA-204 inhibition promoted hCMPC proliferation without affecting cell viability and the level of apoptosis and necrosis, indicating that miRNA-204 might be required for hCMPC differentiation. Quantitative reverse transcriptase-polymerase chain reactions were used to detect the expression profile of cardiac genes, including MEF2C, GATA-4, Nkx-2.5, TropT, βMHC, and cActin. Cardiac α-actin staining was used to quantify the degree of differentiation. MiRNA-204 inhibition significantly down-regulated TropT, βMHC, and cActin and reduced differentiation by 47.81% after 2 weeks of differentiation induction. Interestingly, miRNA-204 mimics (30 nM) did not promote hCMPC proliferation and differentiation. The bioinformatic tool GOmir identified the activating transcription factor 2 (ATF-2) as a potential target, which was confirmed by Western blot and a luciferase reporter assay. ATF-2 overexpression promoted hCMPC proliferation, further demonstrating the role played by ATF-2 as a target gene of miRNA-204. Therefore, miRNA-204 is required for hCMPC differentiation and ATF-2 is a target gene of miRNA-204 in hCMPCs. This study indicates that miRNA-204 is among the regulators that drive hCMPC proliferation and differentiation, and miRNA-204 might be used to influence cell fate.
authors.affiliation: Key Laboratory of Basic Research in Cardiology of Ministry of Education of China, East Hospital, Tongji University School of Medicine, 200120 Shanghai, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Xiao
authors.forename: Dandan
authors.initials: D
authors.lastname: Liang
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Dasheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Pan
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.forename: Pieter A
authors.initials: PA
authors.lastname: Doevendans
authors.forename: Yunfu
authors.initials: Y
authors.lastname: Sun
authors.forename: Xingqun
authors.initials: X
authors.lastname: Liang
authors.forename: Joost P G
authors.initials: JP
authors.lastname: Sluijter
authors.forename: Yi-Han
authors.initials: YH
authors.lastname: Chen
mesh_terms: D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D032386:Myoblasts, Cardiac / Q000166:cytology* / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009336:Necrosis / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D051698:Activating Transcription Factor 2; C549404:MIRN204 microRNA, human; D035683:MicroRNAs