Ye Weihua, Huang Yiyong, Zhu Guanghui, Yan An, Liu Yaoxi, Xiao Han, Mei Haibo
Department of Orthopaedics, Hunan Children's Hospital, Changsha 410007, Hunan Province, PR China.
Regen Ther. 2022 Oct 20;21:477-485. doi: 10.1016/j.reth.2022.09.005. eCollection 2022 Dec.
Congenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear.
The osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter.
CPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8.
miR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT.
先天性胫骨假关节（CPT）是一种罕见的先天性畸形，是骨不连的一种特殊亚型。CPT来源的间充质干细胞（MSCs）成骨分化能力较低可导致CPT进展，而miR-30a可抑制成骨分化。然而，miR-30a在CPT来源的MSCs中的作用仍不清楚。
用miR-30a抑制剂处理CPT来源的MSCs后，通过茜素红S染色和碱性磷酸酶（ALP）活性检测其成骨分化情况。分别通过蛋白质免疫印迹法或定量逆转录-聚合酶链反应（RT-qPCR）评估蛋白质和mRNA的表达水平。通过双荧光素酶报告基因检测法研究miR-30a与HOXD8之间的相互作用。进行染色质免疫沉淀（ChIP）以评估HOXD8与RUNX2启动子之间的结合关系。
CPT来源的MSCs比正常MSCs表现出更低的成骨分化能力。CPT来源的MSCs中miR-30a表达增加，miR-30a下调促进了CPT来源的MSCs的成骨分化。同时，HOXD8是miR-30a的直接靶点，HOXD8可转录激活RUNX2。此外，miR-30a可通过负向调节HOXD8抑制CPT来源的MSCs的成骨分化。
miR-30a通过靶向HOXD8抑制CPT来源MSCs的成骨分化。因此，本研究可能为CPT提供一种新的治疗策略。
