Department of Orthopedics, The Affiliated Hospital of Qingdao University, Qingdao 266555, Shandong, China.
Department of Orthopedics, Heze Municipal Hospital, Heze 274031, Shandong, China.
Mol Immunol. 2019 Oct;114:251-259. doi: 10.1016/j.molimm.2019.07.027. Epub 2019 Aug 6.
Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms.
The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed.
miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression.
miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.
骨折愈合是一个复杂的过程，骨折患者会出现骨不连或再生受损。微小 RNA（miR）-342-5p 是 Notch 下游分子，其在骨折愈合中的作用尚不清楚。本研究旨在探讨 miR-342-5p 在成骨细胞中的功能作用及其潜在机制。
采用实时定量逆转录聚合酶链反应（qRT-PCR）检测 miR-342-5p 在 MC3T3-E1 细胞或 hMSCs 分化过程中的表达。通过细胞计数试剂盒-8（CCK-8）、台盼蓝染色、流式细胞术、Transwell 试验、Western blot 和 qRT-PCR 分别检测异常表达 miR-342-5p 对细胞增殖、凋亡、迁移以及与增殖和成骨分化相关蛋白表达的影响。最后评估 miR-342-5p 的下游因子和靶基因以及丝裂原活化蛋白激酶激酶（MEK）/细胞外信号调节激酶（ERK）通路的参与情况。
在 MC3T3-E1 细胞或 hMSCs 分化过程中，miR-342-5p 水平降低。细胞转染后，miR-342-5p 过表达显著降低细胞活力，诱导细胞凋亡，抑制增殖、迁移和分化，并下调 Bmp7 表达。后续实验表明，Bmp7 敲低可消除 miR-342-5p 抑制对 MC3T3-E1 细胞的影响。此外，COL4A6 和 Bmp2 被预测为 miR-342-5p 的靶基因。最后，miR-342-5p 抑制通过上调 Bmp7 表达增加 MEK 和 ERK 的磷酸化水平。
miR-342-5p 抑制通过调节 Bmp7 激活 MEK/ERK 通路促进成骨细胞的增殖、迁移和分化。
