miR-181b抑制骨髓间充质干细胞成骨分化可能参与青少年特发性脊柱侧弯骨密度降低

miR-181b inhibits osteogenic differentiation of bone marrow-derived mesenchymal stem cells and might be responsible for decreased bone mineral density of adolescent idiopathic scoliosis

目的探讨miR-181b在正常对照和青少年特发性侧弯患者(AIS)骨髓间充质干细胞(BM-MSCs)之间的表达差异,以及其对BM-MSCs成骨分化能力的影响。方法RT-qPCR检测对照和AIS患者MSCs中miR-181b的表达;将miR-181b模拟物、抑制剂以及相应对照瞬时转染BM-MSCs,随后更换为成骨诱导培养基,比较不同处理组BM-MSCs成骨能力的差异;RT-qPCR和Westernblot检测成骨标志基因以及特异性转录因子的表达;碱性磷酸酶(ALP)染色法以及ALP活性分析法来鉴定成骨早期分化;茜素红染色检测细胞外基质的形成情况;慢病毒在BM-MSCs中过表达miR-181b的模拟物和抑制剂后,建立裸鼠异位骨生成模型,通过组织染色检测类骨质的形成。Westernblot检测miR-181b水平的改变对ERK1/2磷酸化的影响。结果与正常对照比,AIS患者BM-MSCs中miR-181b表达显著升高(P<0.01)。过表达miR-181b明显抑制间充质干细胞体外向成骨分化,降低体内异位骨生成(P<0.05);阻断内源性miR-181b可以促进BM-MSCs体外向成骨分化,增强体内类骨质的形成(P<0.05)。其作用机制是,过表达miR-181b抑制ERK1/2的磷酸化,而抑制miR-181b促进ERK1/2的磷酸化(P<0.05)。结论miR-181b抑制间充质干细胞成骨分化,可能与AIS患者骨密度降低有关。

Objective To compare the expression of miR-181 b in bone marrow-derived mesenchymal stem cells(BM-MSCs) between health controls and adolescent idiopathic scoliosis(AIS) patients and to study the role of miR-181 b in osteogenic differentiation of BM-MSCs. Methods The expression of miR-181 b in BM-MSCs from health controls and AIS patients were detected by RT-qPCR. To investigate the function of miR-181 b, BM-MSCs were transfected with miR-181 b mimic, inhibitor or their corresponding controls, and culture medium was thenchanged to osteogenic induction medium. RT-qPCR and Western blot were used to detect the expression of osteogenesis markers and specific transcriptional factors. ALP staining and ALP activity assay were used to detect early ostogenic differentiation. Alizarin red staining was used to determine later osteogenic differentiation. Lentivirus was utilized to overexpress or block miR-181 b in BM-MSCs, and the effects of miR-181 b on bone formation were evaluated in ectopic bone formation model in nude mice. Western blot was used to detect the levels of phosphorylation ERK1/2 after miR-181 b was overexpressed or blocked. Results MiR-181 b was remarkably upregulated in AIS BM-MSCs compared with that of health control. Overexpression of miR-181 b suppressed osteogenic differentiation of BM-MSCs in vitro and impaired ectopic bone formation in vivo. Inhibition of endogenous miR-181 b promoted ostogenic differentiation of BM-MSCs in vitro and enhanced ectopic bone formation of BM-MSCs in vivo. Overexpression of miR-181 b inhibited the phosphorylation of ERK1/2, while blocking miR-181 b elevated the phosphorylation of ERK1/2. Conclusions MiR-181 b suppresses osteogenic differentiation of BM-MSCs and it may contribute to decreased bone mineral density of AIS.