Granulocyte Research Laboratory, Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark
Blood. 2011 Dec 15;118(25):6649-59. doi: 10.1182/blood-2011-03-339978. Epub 2011 Oct 25.
Smad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3'-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1-induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia-derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1-mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.
Smad4 在 TGF-β 途径中很重要，是 TGF-β1 刺激后转录激活和抑制细胞生长所必需的。我们证明，miR-130a 在粒细胞发生过程中差异表达，并靶向 Smad4 mRNA。Smad4 的转录本存在于中性粒细胞成熟的整个过程中，但 Smad4 蛋白在最不成熟的细胞中无法检测到，而 miR-130a 在这些细胞中高度表达。Smad4 mRNA 的 3'-非翻译区中鉴定出两个 miR-130a 结合位点。在 HEK293、A549 和 32Dcl3 细胞中过表达 miR-130a 会抑制 Smad4 蛋白的合成，而不影响 Smad4 mRNA 水平。miR-130a 在粒细胞系中抑制 Smad4 合成会降低其对 TGF-β1 诱导的生长抑制的敏感性。这种效应可以通过反义探针抑制 miR-130a 的活性或表达缺乏 miR-130a 结合位点的 Smad4 mRNA 来逆转。在 t(8;21)(q22;q22)急性髓系白血病衍生细胞系 Kasumi-1 中发现高内源性 miR-130a 和 Smad4 mRNA 水平以及低 Smad4 蛋白表达。当 miR-130a 被反义 RNA 抑制时，Kasumi-1 细胞中 Smad4 蛋白的量增加，并使其对 TGF-β1 介导的细胞生长抑制敏感。我们的数据表明，miR-130a 通过在 TGF-β 途径中结合 Smad4 参与粒细胞细胞周期的调节。
