ABSTRACT

It has been unraveled that microRNAs (miRNAs) played crucial roles in processes of human diseases, while the role of miR-494-5p in intervertebral disc degeneration (IDD) remains scarcely studied. We aimed to investigate the mechanisms of miR-494-5p in IDD with the involvement of tissue inhibitor of metalloproteinase 3 (TIMP3). Expression of miR-494-5p and TIMP3 in IDD clinical specimens was assessed. The IDD models were established by needle punching, which were then injected with low expression of miR-494-5p or TIMP3 overexpression lentivirus to observe their effects on pathology and apoptosis in IDD mice. The nucleus pulposus cells were isolated and, respectively, treated with miR-494-5p inhibitor or TIMP3 overexpression plasmid to determine the viability, apoptosis, and senescence in vitro. Furthermore, the expression of Aggrecan, Col-2, Caveolin-1, and SA-β-gal in nucleus pulposus cells in vitro were measured. The target relation between miR-494-5p and TIMP3 was determined. An increased expression of miR-494-5p and a decreased expression of TIMP3 were found in IDD. Downregulation of miR-494-5p or overexpression of TIMP3 could relieve pathology and suppress nucleus pulposus cell apoptosis in IDD mice, as well as promote the viability and attenuate the apoptosis and senescence of nucleus pulposus cells from IDD mice. Moreover, inhibition of miR-494-5p or overexpression of TIMP3 upregulated Aggrecan and Col-2 expression while downregulated Caveolin-1 and SA-β-gal expression, and TIMP3 was the target gene of miR-494-5p. Results of this study indicated that the degradation of miR-494-5p ameliorates the development of IDD by elevating TIPM3, which may provide new targets for IDD treatment.

摘要

microRNAs (miRNAs) 在人类疾病的进程中发挥着至关重要的作用，而 miR-494-5p 在椎间盘退变 (IDD) 中的作用却鲜有研究。我们旨在研究 miR-494-5p 在 IDD 中的机制，其中涉及金属蛋白酶 3 组织抑制剂 (TIMP3)。评估了 IDD 临床标本中 miR-494-5p 和 TIMP3 的表达。采用针刺法建立IDD模型，注射低表达miR-494-5p或过表达TIMP3慢病毒，观察其对IDD小鼠病理和凋亡的影响。分离髓核细胞，分别用 miR-494-5p 抑制剂或 TIMP3 过表达质粒处理，以确定体外的活力、凋亡和衰老. 此外, Aggrecan、Col-2、Caveolin-1 和 SA-β-gal在体外髓核细胞中的表达被测量了。确定了 miR-494-5p 和 TIMP3 之间的靶标关系。在 IDD 中发现 miR-494-5p 的表达增加和 TIMP3 的表达减少。下调 miR-494-5p 或过表达 TIMP3 可以缓解 IDD 小鼠的病理和抑制髓核细胞凋亡，并促进 IDD 小鼠髓核细胞的活力并减弱其凋亡和衰老。此外，抑制 miR-494-5p 或过表达 TIMP3 上调 Aggrecan 和 Col-2 表达，同时下调 Caveolin-1 和 SA-β-gal 表达，TIMP3 是 miR-494-5p 的靶基因。本研究结果表明，miR-494-5p 的降解通过提高 TIPM3 来改善 IDD 的发展，这可能为 IDD 治疗提供新的靶点。