Department of Orthopedic, the 309th Hospital of PLA, Beijing 100091, China.
Department of Orthopedic, the 309th Hospital of PLA, Beijing 100091, China.
Life Sci. 2018 Jun 1;202:11-20. doi: 10.1016/j.lfs.2018.03.042. Epub 2018 Mar 22.
MicroRNAs (miRs) play an important role in osteoclastogenesis. However, no study has investigated the underlying molecular mechanisms of miR-145 in this process. The purpose of the present study was to investigate the role of miR-145 and its post-transcriptional mechanism in the progression of osteoclast differentiation.
Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) were used to induce osteoclastogenesis originated from bone marrow-derived macrophages (BMMs). Female C57BL/6J mice were divided into sham, OVX, OVX + NC-agomir and OVX + miR-145-agomir groups. Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify osteoclasts in-vitro and in-vivo. The mRNA and protein levels in osteoclast and tibia were assayed by qRT-PCR and western blotting, respectively.
miR-145 expression was inhibited in RANKL-induced osteoclastogenesis, whereas overexpression of miR-145 attenuated it. We further found that Smad3 is a direct target gene of miR-145 by binding with its 3'-UTR. Overexpression of miR-145 significantly suppressed Smad3 mRNA and protein expression. In-vivo, miR-145 agomir treatment inhibited osteoclast activity in OVX mice by inhibiting Smad3 expression.
We provide the evidence that over-expression of miR-145 could inhibit osteoclast differentiation, at least partially, by decreasing Smad3 expression.
微小 RNA（miRs）在破骨细胞分化中发挥重要作用。然而，目前尚无研究探讨 miR-145 在这一过程中的潜在分子机制。本研究旨在探讨 miR-145 在破骨细胞分化进展中的作用及其转录后机制。
巨噬细胞集落刺激因子（M-CSF）和核因子κB 受体激活配体（RANKL）用于诱导骨髓来源巨噬细胞（BMMs）来源的破骨细胞分化。雌性 C57BL/6J 小鼠分为假手术组、去卵巢组、去卵巢+阴性对照 agomir 组和去卵巢+miR-145 agomir 组。体外和体内通过抗酒石酸酸性磷酸酶（TRAP）染色鉴定破骨细胞。通过 qRT-PCR 和 Western blot 分别检测破骨细胞和胫骨中的 mRNA 和蛋白水平。
miR-145 在 RANKL 诱导的破骨细胞分化中受到抑制，而过表达 miR-145 则减弱了这种抑制作用。我们进一步发现 Smad3 是 miR-145 的直接靶基因，通过与 3'-UTR 结合。miR-145 的过表达显著抑制了 Smad3 的 mRNA 和蛋白表达。在体内，miR-145 agomir 处理通过抑制 Smad3 表达抑制了 OVX 小鼠的破骨细胞活性。
我们提供的证据表明，通过降低 Smad3 表达，过表达 miR-145 可部分抑制破骨细胞分化。
