Department of Orthopedics, The Second Affiliated Hospital of Jiaxing University, No. 1518 Huanchengbei Road, Jiaxing, Zhejiang, 314299, China.
J Orthop Surg Res. 2020 Jun 29;15(1):237. doi: 10.1186/s13018-020-01685-8.
Atrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. microRNA dysregulation may underlie the dysfunctional osteogenesis in atrophic non-union fractures. Here, we aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323's underlying mechanism of action in human mesenchymal stromal cells.
Human atrophic non-union and standard healing fracture specimens were examined using H&E and Alcian Blue staining, immunohistochemistry, qRT-PCR, immunoblotting, and ALP activity assays. The effects of miR-1323 mimics or inhibition on BMP4, SMAD4, osteogenesis-related proteins, ALP activity, and bone mineralization were analyzed in human mesenchymal stromal cells. Luciferase reporter assays were utilized to assay miR-1323's binding to the 3'UTRs of BMP4 and SMAD4. The effects of miR-1323, BMP4, and SMAD4 were analyzed by siRNA and overexpression vectors. A rat femur fracture model was established to analyze the in vivo effects of antagomiR-1323 treatment.
miR-1323 was upregulated in human atrophic non-union fractures. Atrophic non-union was associated with downregulation of BMP4 and SMAD4 as well as the osteogenic markers ALP, collagen I, and RUNX2. In vitro, miR-1323 suppressed BMP4 and SMAD4 expression by binding to the 3'UTRs of BMP4 and SMAD4. Moreover, miR-1323's inhibition of BMP4 and SMAD4 inhibited mesenchymal stromal cell osteogenic differentiation via modulating the nuclear translocation of the transcriptional co-activator TAZ. In vivo, antagomiR-1323 therapy facilitated the healing of fractures in a rat model of femoral fracture.
This evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.
萎缩性骨不连骨折在骨折后 3 个月内没有骨痂形成的放射学证据。miRNA 失调可能是萎缩性骨不连骨折骨生成功能障碍的基础。在这里，我们旨在分析人类萎缩性骨不连骨折中 miR-1323 的表达，并研究 miR-1323 在人间充质基质细胞中的作用机制。
使用 H&E 和阿尔辛蓝染色、免疫组织化学、qRT-PCR、免疫印迹和 ALP 活性测定法检查人类萎缩性骨不连和标准愈合骨折标本。分析 miR-1323 模拟物或抑制物对人骨髓基质细胞中 BMP4、SMAD4、成骨相关蛋白、ALP 活性和骨矿化的影响。利用荧光素酶报告基因测定法检测 miR-1323 与 BMP4 和 SMAD4 的 3'UTR 的结合。通过 siRNA 和过表达载体分析 miR-1323、BMP4 和 SMAD4 的作用。建立大鼠股骨骨折模型分析 antagomiR-1323 治疗的体内作用。
miR-1323 在人类萎缩性骨不连骨折中上调。萎缩性骨不连与 BMP4 和 SMAD4 以及成骨标志物 ALP、胶原 I 和 RUNX2 的下调有关。在体外，miR-1323 通过与 BMP4 和 SMAD4 的 3'UTR 结合抑制 BMP4 和 SMAD4 的表达。此外，miR-1323 对 BMP4 和 SMAD4 的抑制通过调节转录共激活因子 TAZ 的核易位抑制间充质基质细胞成骨分化。在体内，antagomiR-1323 治疗促进了大鼠股骨骨折模型骨折愈合。
该证据支持 miR-1323/BMP4 和 miR-1323/SMAD4 轴作为萎缩性骨不连骨折的新治疗靶点。
