Zhao Huiwu, Kalota Anna, Jin Shenghao, Gewirtz Alan M
Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Blood. 2009 Jan 15;113(3):505-16. doi: 10.1182/blood-2008-01-136218. Epub 2008 Sep 25.
The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34(+) cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.
c-myb原癌基因编码一种对谱系定向、增殖和分化至关重要的造血细胞特异性转录因子。鉴于其关键功能，c-Myb调节因子备受关注，但仍未完全明确。在此我们表明，c-Myb的表达受到微小RNA（miRNA）-15a的转录后调控。通过荧光素酶报告基因检测，我们发现miR-15a直接结合c-myb mRNA的3'-UTR。用miR-15a模拟物转染K562髓系白血病细胞，证明了结合的功能性。该模拟物降低了c-Myb的表达，并使细胞停滞在细胞周期的G(1)期。缺乏3'-UTR的c-myb mRNA的外源性表达部分挽救了miR-15a诱导的细胞周期阻滞。有趣的是，miR-15a启动子包含几个潜在的c-Myb蛋白结合位点。通过染色质免疫沉淀分析证实了一个典型c-Myb结合位点的占据，并表明其是K562细胞中miR-15a表达所必需的。最后，在使用正常人CD34(+)细胞的研究中，我们表明在经历红系分化的细胞中，c-Myb和miR-15a的表达呈负相关，并且miR-15a的过表达在体外阻断了红系和髓系集落的形成。总的来说，这些发现表明存在一个在人类造血过程中可能具有重要意义的c-Myb-miR-15a自动调节反馈环。
