Abstract
Phorbol 12-myristate 13-acetate (PMA) induces megakaryocytic differentiation of the human chronic myelocytic leukemia cell line K562. We examined the potential regulatory role of microRNAs (miRNAs) in this process. Genome-wide expression profiling identified 21 miRNAs (miRs) that were induced by the treatment of K562 cells with PMA. Among them, the expression of miR-34a, miR-221, and miR-222 was induced in the early stages and maintained throughout the late stages of differentiation. Cell signaling analysis showed that the activation of extracellular signal-regulated protein kinase (ERK) in response to PMA strongly induced miR-34a expression by transactivation via the activator protein-1 binding site in the upstream region of the miR-34a gene. Reporter gene assays identified mitogen-activated protein kinase kinase 1 (MEK1) as a direct target of miR-34a and c-fos as a direct target of miR-221/222. Although overexpression of the three miRNAs had little effect on cell differentiation, overexpression of miR-34a significantly repressed the proliferation of K562 cells with a concomitant reduction in MEK1 protein expression. Conversely, a locked nucleic acid probe against miR-34a significantly enhanced the proliferation of PMA-treated K562 cells. Taken together, the results show that PMA activates the MEK-ERK pathway and strongly induces miRNA-34a expression, which in turn inhibits cell proliferation by repressing the expression of MEK1. Thus, the results highlight an important regulatory role for miR-34a in the process of megakaryocytic differentiation, especially in the arrest of cell growth, which is a prerequisite for cells to enter differentiation.
Footnotes
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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This work was supported in part by Grants-in-Aid for Scientific Research (S) [Grant 19109001], the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, the Japan Health Science Foundation, the Ministry of Human Health and Welfare, the Mitsubishi Foundation, the Uehara Memorial Foundation, the Sankyo Foundation of Life Science, and Grants-in-Aid for the Japan Society for the Promotion of Science Fellows [Grant 09J03338].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.063321.
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ABBREVIATIONS:
- PMA
- phorbol 12-myristate 13-acetate
- MEK
- mitogen-activated protein kinase kinase
- PKC
- protein kinase C
- miRNA (miR)
- microRNA
- ERK
- extracellular signal-regulated protein kinase
- p-ERK
- phospho-ERK
- MAPK
- mitogen-activated protein kinase
- UTR
- untranslated region
- DMSO
- dimethyl sulfoxide
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- qRT-PCR
- quantitative real-time PCR
- 5′ RACE
- rapid amplification of 5′-cDNA ends
- LNA
- locked nucleic acid
- U0126
- 1.4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene
- ANOVA
- analysis of variance
- AP-1
- activator protein-1
- Ct
- threshold cycle
- snRNA
- small nuclear RNA
- NC
- negative control.
- Received December 29, 2009.
- Accepted March 18, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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