lncTIMP3 通过 miR-214/Smad4 轴促进骨髓间充质干细胞的成骨分化，从而缓解绝经后骨质疏松症。
lncTIMP3 promotes osteogenic differentiation of bone marrow mesenchymal stem cells via miR-214/Smad4 axis to relieve postmenopausal osteoporosis.
机构信息
Laboratory of New Techniques of Restoration & Reconstruction, Institute of Traumatology & Orthopedics, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China.
School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210023, Jiangsu, China.
出版信息
Mol Biol Rep. 2024 Jun 1;51(1):719. doi: 10.1007/s11033-024-09652-w.
BACKGROUND
Promoting the balance between bone formation and bone resorption is the main therapeutic goal for postmenopausal osteoporosis (PMOP), and bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation plays an important regulatory role in this process. Recently, several long non-coding RNAs (lncRNAs) have been reported to play an important regulatory role in the occurrence and development of OP and participates in a variety of physiological and pathological processes. However, the role of lncRNA tissue inhibitor of metalloproteinases 3 (lncTIMP3) remains to be investigated.
METHODS
The characteristics of BMSCs isolated from the PMOP rat model were verified by flow cytometry assay, alkaline phosphatase (ALP), alizarin red and Oil Red O staining assays. Micro-CT and HE staining assays were performed to examine histological changes of the vertebral trabeculae of the rats. RT-qPCR and western blotting assays were carried out to measure the RNA and protein expression levels. The subcellular location of lncTIMP3 was analyzed by FISH assay. The targeting relationships were verified by luciferase reporter assay and RNA pull-down assay.
RESULTS
The trabecular spacing was increased in the PMOP rats, while ALP activity and the expression levels of Runx2, Col1a1 and Ocn were all markedly decreased. Among the RNA sequencing results of the clinical samples, lncTIMP3 was the most downregulated differentially expressed lncRNA, also its level was significantly reduced in the OVX rats. Knockdown of lncTIMP3 inhibited osteogenesis of BMSCs, whereas overexpression of lncTIMP3 exhibited the reverse results. Subsequently, lncTIMP3 was confirmed to be located in the cytoplasm of BMSCs, implying its potential as a competing endogenous RNA for miRNAs. Finally, the negative targeting correlations of miR-214 between lncTIMP3 and Smad4 were elucidated in vitro.
CONCLUSION
lncTIMP3 may delay the progress of PMOP by promoting the activity of BMSC, the level of osteogenic differentiation marker gene and the formation of calcium nodules by acting on the miR-214/Smad4 axis. This finding may offer valuable insights into the possible management of PMOP.
背景
促进骨形成和骨吸收之间的平衡是绝经后骨质疏松症（PMOP）的主要治疗目标，骨髓间充质干细胞（BMSCs）成骨分化在这一过程中起着重要的调节作用。最近，有几种长链非编码 RNA（lncRNA）被报道在 OP 的发生和发展中起着重要的调节作用，并参与各种生理和病理过程。然而，lncRNA 组织金属蛋白酶抑制剂 3（lncTIMP3）的作用仍有待研究。
方法
通过流式细胞术、碱性磷酸酶（ALP）、茜素红和油红 O 染色试验验证从 PMOP 大鼠模型中分离的 BMSCs 的特征。通过微 CT 和 HE 染色试验检查大鼠椎骨小梁的组织学变化。通过 RT-qPCR 和 Western blot 试验测定 RNA 和蛋白质表达水平。通过 FISH 试验分析 lncTIMP3 的亚细胞定位。通过荧光素酶报告试验和 RNA 下拉试验验证靶向关系。
结果
PMOP 大鼠的骨小梁间距增加，而 ALP 活性以及 Runx2、Col1a1 和 Ocn 的表达水平均显著降低。在临床样本的 RNA 测序结果中，lncTIMP3 是下调最明显的差异表达 lncRNA，其在 OVX 大鼠中的水平也显著降低。lncTIMP3 的敲低抑制了 BMSCs 的成骨作用，而过表达 lncTIMP3 则呈现相反的结果。随后，证实 lncTIMP3 位于 BMSCs 的细胞质中，表明其可能作为 miRNA 的竞争内源性 RNA。最后，在体外阐明了 lncTIMP3 与 Smad4 之间的 miR-214 的负靶向关系。
结论
lncTIMP3 可能通过作用于 miR-214/Smad4 轴，促进 BMSC 活性、成骨分化标志物基因水平和钙结节形成，从而延缓 PMOP 的进展。这一发现可能为 PMOP 的可能治疗提供有价值的见解。
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