Crosstalk between microRNA-21-5p and the transcription factor Dec1 maintains osteoblast function
Crosstalk between microRNA-21-5p and the transcription factor Dec1 maintains osteoblast function
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DOI：
10.1016/j.bbrc.2022.09.090
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发表时间：
2022-10-01
影响因子：
3.1
通讯作者：
Bhawal, Ujjal K.
中科院分区：
文献类型：
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作者：
Kurita, Takashi;Li, Xiaoyan;Bhawal, Ujjal K.
MicroRNA 与骨形成中关键的转录后基因调控相关。人分化胚胎软骨细胞表达基因 1 (Dec1) 也参与调节成骨细胞发生。在本研究中，我们旨在研究 miR-21-5p 和 Dec1 在成骨细胞功能中的独特作用并确定其生物学功能。 MC3T3-E1 前成骨细胞用于体外分析。使用 miR-21-5p 敲除 (KO) 小鼠、Dec1KO 小鼠和年龄匹配的野生型 (WT) 小鼠来表征 miR-21-5p 和 Dec1 缺陷对骨形成的影响。进行形态学分析 [微型计算机断层扫描 (micro-CT)]，并收集测量结果以验证 miR-21-5pKO 小鼠。通过H-E染色、阿赞染色、马森三色染色和甲苯胺蓝染色评估小鼠股骨组织的组织病理学变化。使用定量实时 RT-PCR、蛋白质印迹和免疫组织化学染色来表征碱性磷酸酶、Runx2、Osterix、骨桥蛋白、Dec1 和 miR-21-5p 的表达水平。进行生物信息学分析和双荧光素酶报告基因测定，以确认 Dec1 是 miR-21-5p 的靶标。 Dec1 表达从成骨细胞诱导的第 7 天开始逐渐增加，而 miR-21-5p 在第 21 天出现峰值。在非诱导的成骨细胞中，使用 miR-21-5p 模拟物进行机械功能获得转染研究增强了 Runx2 和 Osterix 的表达，但抑制了 Dec1。 miR-21-5pKO 小鼠骨骼生长减少。与 WT 小鼠相比，Dec1 缺陷小鼠在 12 周龄时表现出先进的骨形成。 Dec1 缺陷上调了 Dec1KO 小鼠股骨中 Runx2 和 Osterix 的表达。然而，与 WT 小鼠股骨相比，这些变化在 miR-21-5pKO 小鼠股骨中发生逆转。双荧光素酶报告基因检测表明 Dec1 可能是 miR-21-5p 的下游靶标。这些发现表明，由于 miR-21-5p 缺陷导致的成骨潜力降低是通过增强 Dec1 表达来实现的，并且 miR-21-5p/Dec1 轴参与调节成骨细胞功能。(c) 2022 Elsevier Inc. 保留所有权利。
MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteo-blastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21-5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21-5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21-5p and Dec1 deficiencies on bone for-mation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and mea-surements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21-5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21-5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21-5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of -function transfection study with a miR-21-5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21-5p. These findings showed that the reduced osteogenic potential due to a miR-21-5p deficiency is achieved by enhanced Dec1 expression and that the miR-21-5p/Dec1 axis is involved in regulating osteoblast function.(c) 2022 Elsevier Inc. All rights reserved.