Role of miR-222-3p in c-Src-Mediated Regulation of Osteoclastogenesis.
Role of miR-222-3p in c-Src-Mediated Regulation of Osteoclastogenesis.
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DOI：
10.3390/ijms17020240
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发表时间：
2016-02-16
影响因子：
5.6
通讯作者：
Hamamura K
中科院分区：
文献类型：
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作者：
Takigawa S;Chen A;Wan Q;Na S;Sudo A;Yokota H;Hamamura K
MicroRNAs(MiRNAs)是一种小的非编码RNA，主要在基因表达中发挥转录后调节作用。利用RAW264.7破骨前细胞和全基因组表达分析，我们鉴定了一组参与破骨细胞形成的miRNAs。在计算机分析的基础上，我们专门针对miR-222-3p进行了研究，并评价了其在破骨细胞形成中的作用。结果表明，miR-222-3p抑制剂上调活化的T细胞核因子、胞浆1(NFATc1)和抗酒石酸酸性磷酸酶(TRAP)的mRNA水平，而其模拟剂下调它们的mRNA水平。Western印迹分析显示，其抑制剂使TRAP和组织蛋白酶K的蛋白水平升高，而其模拟剂降低了它们的水平。在存在和不存在核因子受体激活剂κ-B配体(RANKL)的情况下，全基因组的基因表达分析预测c-Src是miR-222-3p的一个潜在的调控靶点。采用荧光共振能量转移(FRET)技术的活细胞成像显示miR-222-3p对c-Src活性具有抑制作用，c-Src的部分沉默抑制了RANKL诱导的TRAP和组织蛋白酶K的表达，以及多核破骨细胞的数量及其凹陷的形成。综上所述，本研究证明miR-222-3p是破骨细胞生成的抑制因子，c-Src介导其对组织蛋白酶K和TRAP的抑制。
MicroRNAs (miRNAs) are small non-coding RNAs that play a mostly post-transcriptional regulatory role in gene expression. Using RAW264.7 pre-osteoclast cells and genome-wide expression analysis, we identified a set of miRNAs that are involved in osteoclastogenesis. Based on in silico analysis, we specifically focused on miR-222-3p and evaluated its role in osteoclastogenesis. The results show that the inhibitor of miR-222-3p upregulated the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP), while its mimicking agent downregulated their mRNA levels. Western blot analysis showed that its inhibitor increased the protein levels of TRAP and cathepsin K, while its mimicking agent decreased their levels. Genome-wide mRNA expression analysis in the presence and absence of receptor activator of nuclear factor κ-B ligand (RANKL) predicted c-Src as a potential regulatory target of miR-222-3p. Live cell imaging using a fluorescence resonance energy transfer (FRET) technique revealed that miR-222-3p acted as an inhibitor of c-Src activity, and a partial silencing of c-Src suppressed RANKL-induced expression of TRAP and cathepsin K, as well as the number of multi-nucleated osteoclasts and their pit formation. Collectively, the study herein demonstrates that miR-222-3p serves as an inhibitor of osteoclastogenesis and c-Src mediates its inhibition of cathepsin K and TRAP.