mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk

IF 4.3 3区 材料科学 Q1 ENGINEERING, ELECTRICAL & ELECTRONIC ACS Applied Electronic Materials Pub Date : 2024-05-01 DOI:10.1002/jgm.3687
Dan He, Yueying Jiao, Jian Xu, Junjie Luo, Yaqi Cui, Xiabing Han, Hongshan Zhao
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Abstract

Background

Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration.

Methods

Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p.

Results

miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk.

Conclusions

miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

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mmu-miR-185 通过靶向 Btk 调节破骨细胞的分化和迁移
背景骨骼不断重塑,这一过程涉及破骨细胞介导的骨吸收和成骨细胞介导的骨形成,对维持健康的骨量至关重要。我们以前曾观察到,miR-185 的消耗可通过调节 Bgn 的表达和 BMP/Smad 信号通路来促进骨形成。然而,miR-185-5p 对破骨细胞和骨重塑的影响尚未阐明,值得进一步探讨。 方法 利用耐酒石酸磷酸酶染色法评估 mmu-miR-185 基因敲除(KO)小鼠和野生型(WT)小鼠骨髓单核巨噬细胞(BMMs)的分化能力。反转录酶定量 PCR 比较了 KO 组和 WT 组骨髓单核巨噬细胞中 miR-185-5p 和破骨细胞标记分子(包括 Trap、Dcstamp、Ctsk 和 Nfatc1)的差异。采用 Western 印迹分析观察破骨细胞标记分子的表达。使用细胞计数试剂盒-8分析细胞增殖能力。透孔实验用于检测细胞迁移。采用双荧光素酶报告实验证实 Btk 是否是 miR-185-5p 的下游靶基因。 结果 miR-185 的缺失促进了骨髓单核细胞/巨噬细胞的破骨细胞分化。在 RAW264.7 细胞中过表达 miR-185-5p 可抑制破骨细胞的分化和迁移。此外,Btk 被确定为 miR-185-5p 的下游靶基因,这表明 miR-185-5p 可能通过靶向 Btk 来抑制破骨细胞的分化和迁移。 结论 miR-185 可调控破骨细胞的分化,过表达 miR-185-5p 可抑制体外破骨细胞的分化和迁移。此外,miR-185-5p 可能通过调节 Btk 的表达来调节破骨细胞的分化和迁移。
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7.20
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4.30%
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567
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