State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;
Second Hospital of Hebei Medical University, Shijiazhuang, Hebei Province, China;
J Leukoc Biol. 2014 Dec;96(6):1023-35. doi: 10.1189/jlb.1A0514-240R. Epub 2014 Sep 25.
miRNAs are short, noncoding RNAs that regulate expression of target genes at post-transcriptional levels and function in many important cellular processes, including differentiation, proliferation, etc. In this study, we observed down-regulation of miR-199a-5p during monocyte/macrophage differentiation of HL-60 and THP-1 cells, as well as human CD34(+) HSPCs. This down-regulation of miR-199a-5p resulted from the up-regulation of PU.1 that was demonstrated to regulate transcription of the miR-199a-2 gene negatively. Overexpression of miR-199a-5p by miR-199a-5p mimic transfection or lentivirus-mediated gene transfer significantly inhibited monocyte/macrophage differentiation of the cell lines or HSPCs. The mRNA encoding an ACVR1B was identified as a direct target of miR-199a-5p. Gradually increased ACVR1B expression level was detected during monocyte/macrophage differentiation of the leukemic cell lines and HSPCs, and knockdown of ACVR1B resulted in inhibition of monocyte/macrophage differentiation of HL-60 and THP-1 cells, which suggested that ACVR1B functions as a positive regulator of monocyte/macrophage differentiation. We demonstrated that miR-199a-5p overexpression or ACVR1B knockdown promoted proliferation of THP-1 cells through increasing phosphorylation of Rb. We also demonstrated that the down-regulation of ACVR1B reduced p-Smad2/3, which resulted in decreased expression of C/EBPα, a key regulator of monocyte/macrophage differentiation, and finally, inhibited monocyte/macrophage differentiation.
miRNAs 是短的非编码 RNA，在转录后水平上调节靶基因的表达，在许多重要的细胞过程中发挥作用，包括分化、增殖等。在这项研究中，我们观察到 miR-199a-5p 在 HL-60 和 THP-1 细胞以及人 CD34(+) HSPCs 的单核细胞/巨噬细胞分化过程中下调。miR-199a-5p 的这种下调是由于 PU.1 的上调引起的，PU.1 被证明负调控 miR-199a-2 基因的转录。通过 miR-199a-5p 模拟物转染或慢病毒介导的基因转移过表达 miR-199a-5p 可显著抑制细胞系或 HSPCs 的单核细胞/巨噬细胞分化。编码 ACVR1B 的 mRNA 被鉴定为 miR-199a-5p 的直接靶标。在白血病细胞系和 HSPCs 的单核细胞/巨噬细胞分化过程中，逐渐检测到 ACVR1B 表达水平增加，并且敲低 ACVR1B 导致 HL-60 和 THP-1 细胞的单核细胞/巨噬细胞分化受到抑制，这表明 ACVR1B 作为单核细胞/巨噬细胞分化的正调节剂发挥作用。我们证明 miR-199a-5p 过表达或 ACVR1B 敲低通过增加 Rb 的磷酸化促进 THP-1 细胞的增殖。我们还证明，ACVR1B 的下调降低了 p-Smad2/3，导致单核细胞/巨噬细胞分化的关键调节因子 C/EBPα 的表达减少，最终抑制单核细胞/巨噬细胞分化。
