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Identification of microRNAs regulated by activin A in human embryonic stem cells§

Authors

  • Zong-Yun Tsai,

    1. Department of Medicinal and Applied Chemistry, College of Life Sciences, Kaohsiung Medical University, Kaohsiung 807, Taiwan
    2. Stem Cell Laboratory, Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
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  • Sher Singh,

    1. Department of Life Science, College of Science, National Taiwan Normal University, Taipei 116, Taiwan
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  • Sung-Liang Yu,

    1. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
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  • Li-Pin Kao,

    1. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
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  • Bo-Zhi Chen,

    1. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
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  • Bing-Ching Ho,

    1. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei 100, Taiwan
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  • Pan-Chyr Yang,

    1. Department of Internal Medicine, National Taiwan University College of Medicine, Taipei 100, Taiwan
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  • Steven Shoei-Lung Li

    Corresponding author
    1. Stem Cell Laboratory, Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
    2. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
    • Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
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  • Authors' contributions: ZYT cultured and characterized T3MF, T3CM, and T3BA cells, as well as did part of data analyses. SS did bioinformatic analyses. S-LY supervised the determination of both miRNAs and mRNAs. L-PK prepared RNA samples. BZC maintained T3MF cells. BCH did luciferase assay. PCY directed the Microarray Core Facility for Genomic Medicine of National Taiwan University College of Medicine where the analyses of miRNAs and mRNAs were done. SSLL designed the experiments, analyzed results, and wrote the manuscript.

  • Database and accession number: The original data obtained from Affymetrix human genome U133 plus 2.0 GeneChip have been deposited to NCBI database, and the GEO series number is GSE16910.

  • §

    Zong-Yun Tsai and Sher Singh contributed equally to this work.

  • The authors declare no competing financial interests.

Abstract

Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367, and miR-200c, as well as three other microRNAs miR-199a, miR-19a, and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b, and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24, and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4, and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells. J. Cell. Biochem. 109: 93–102, 2010. © 2009 Wiley-Liss, Inc.

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