Department of Spine Surgery, The First Affiliated Hospital of Dali University, No.32, Jiashibo Avenue, Dali, 671000, Yunnan Province, People's Republic of China.
Mol Med. 2021 Apr 26;27(1):43. doi: 10.1186/s10020-021-00303-5.
Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown.
To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p.
The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation.
MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.
骨质疏松症严重扰乱了人们的生活。同时，成骨分化的抑制或减弱是骨质疏松症发病机制的重要因素之一。有报道称，miR-27a-3p 可减轻骨质疏松症的症状。然而，miR-27a-3p 在成骨分化中的作用机制在很大程度上尚不清楚。
用成骨诱导培养基（OIM）诱导 MC3T3-E1 细胞的成骨分化。用 RT-qPCR 评估细胞中 miR-27a-3p 和 CRY2 的 mRNA 表达。用 Western blot 检测 MC3T3-E1 细胞中 CRY2、Runt 相关转录因子 2（Runx2）、骨桥蛋白（OPN）、骨钙素（OCN）的蛋白水平以及细胞外调节蛋白激酶（ERK）1/2 的磷酸化水平。同时，通过茜素红染色和 ALP 试剂盒分别检测钙结节和 ALP 活性。用荧光素酶报告基因检测分析 CRY2 和 miR-27a-3p 之间的相关性。
OIM 可增加 MC3T3-E1 细胞中 miR-27a-3p 的表达和 ERK1/2 的磷酸化水平，同时降低 CRY2 的表达。此外，下调 miR-27a-3p 和过表达 CRY2 可显著逆转 OIM 诱导的钙化结节、ALP 含量和骨形成相关蛋白表达的增加。此外，miR-27a-3p 可直接靶向 CRY2，并负调控 CRY2。同时，miR-27a-3p 抑制剂对 OIM 处理的 MC3T3-E1 细胞的成骨分化的抑制作用可被 CRY2 的敲低或 honokiol（ERK1/2 信号激活剂）所逆转。此外，miR-27a-3p 可显著抑制 OIM 处理的 MC3T3-E1 细胞的凋亡。总之，miR-27a-3p/CRY2/ERK 轴在成骨细胞分化中起重要作用。
miR-27a-3p 通过介导 CRY2/ERK1/2 轴促进成骨细胞分化。因此，miR-27a-3p 可能成为治疗骨质疏松症的新靶点。
