Thrombopoietin regulates c-Myb expression by modulating micro RNA 150 expression.
Thrombopoietin regulates c-Myb expression by modulating micro RNA 150 expression.
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DOI：
10.1016/j.exphem.2008.07.001
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发表时间：
2008-12
影响因子：
2.6
通讯作者：
Kaushansky, Kenneth
中科院分区：
文献类型：
--
作者：
Barroga, Charlene F.;Pham, Hang;Kaushansky, Kenneth
由于巨核细胞及其祖细胞数量的增加，携带c-Myb亚型突变的小鼠表现出增强的血小板生成。血小板生成素诱导这些相同的作用，这使我们假设激素通过调节c-Myb表达起作用，因为c-Myb水平在血小板生成素诱导的MK成熟期间福尔斯下降。微小RNA（miRs）通过与特定mRNA的3′非翻译区（UTR）结合来下调基因表达;我们注意到c-Myb的3′UTR含有4个miR-150结合位点。我们使用定量RT-PCR、蛋白质印迹和报告基因分析来评估c-Myb对血小板生成素刺激的反应以及miR-150表达的增加和减少。我们发现血小板生成素在7小时内降低了巨核细胞和UT 7/TPO细胞中c-Myb mRNA和蛋白水平。使用包含c-Myb 3′UTR区域（包括4个miR 150结合位点）的报告基因，我们发现在UT 7/TPO细胞中，miR 150的表达使荧光素酶表达在24 h时降至基线的50%，在48 h时降至25%。定量PCR和蛋白质印迹也显示，miR-150在UT 7/TPO细胞中将内源性c-Myb mRNA和蛋白质降低至50%，在成熟巨核细胞中降低至65%。利用抗miR 150的匡威实验使萤光素酶活性增加至对照抗miR的2倍。最后，促血小板生成素在24小时内使miR 150表达增加1.8倍，在48小时内增加3.4倍。这些发现证实miR 150下调c-Myb水平，并且由于促血小板生成素影响miR 150表达，我们的结果表明，除了影响MK祖细胞生长外，促血小板生成素通过诱导miR-150下调c-Myb表达。
Mice harboring c-Myb hypomorphic mutations display enhanced thrombopoiesis because of increased numbers of megakaryocytes and their progenitors. Thrombopoietin induces these same effects, which lead us to hypothesize that the hormone acts through modulation of c-Myb expression, as c-Myb levels falls during thrombopoietin-induced MK maturation. Micro RNAs (miRs) down-regulate gene expression by binding to the 3′ untranslated region (UTR) of specific mRNAs; we noted that the 3′UTR of c-Myb contains four miR-150 binding sites. We used quantitative RT-PCR, western blotting and reporter gene analyses to assess the response of c-Myb to thrombopoietin stimulation and to gain of- and loss of-miR-150 expression. We found that thrombopoietin reduced c-Myb mRNA and protein levels within 7 hr in megakaryocytes and UT7/TPO cells. Using a reporter gene containing the c-Myb 3′UTR region, including its 4 miR150 binding sites, we found that expression of miR150 reduced luciferase expression to 50% of baseline at 24 hr and to 25% at 48 hr in UT7/TPO cells. Quantitative-PCR and western blotting also revealed that miR-150 reduced endogenous c-Myb mRNA and protein to 50% in UT7/TPO cells, and to 65% in mature megakaryocytes. Converse experiments utilizing anti-miR150 increased luciferase activity 2-fold over control anti-miR. Finally, thrombopoietin increased miR150 expression 1.8-fold within 24 hr and 3.4-fold within 48 hr. These findings establish that miR150 down-modulates c-Myb levels, and since thrombopoietin affects miR150 expression, our results indicate that in addition to affecting MK progenitor cell growth, thrombopoietin down-modulates c-Myb expression through the induction of miR-150.
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