ABSTRACT miRNAs play an important role in the pathogenesis of intervertebral disc degeneration (IDD). The role and the underlying mechanism of miR-424-5p in human nucleus pulposus (NP) are still unknown. We aimed to explore the role of miR-424-5p in IDD. Real-time PCR was used to detect the expression of miR-424-5p and Bcl2 in IDD tissues and idiopathic scoliosis tissues. Human NP cells were used in our study. MTT and Hoechst apoptosis assays were used to detect the proliferation and apoptosis of NP cells, respectively. Western blotting assays were used to detect the expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells. A luciferase reporter assay was applied to confirm the relationship between miR-424-5p and Bcl2. Our results showed that the expression of miR-424-5p was increased and Bcl2 was decreased in degenerative NP cells. miR-425-5p expression was negatively correlated with Bcl2 expression in IDD tissues. Suppression of miR-424-5p using an inhibitor increased Bcl2 expression at both the mRNA and protein levels, and it promoted cell viability and inhibited apoptosis. Furthermore, the levels of cleaved caspase-3 and cleaved caspase-9 were downregulated in miR-424-5p-silenced NP cells. Interestingly, we found that silencing miR-424-5p increased p62 expression at both the mRNA and protein levels. Finally, a luciferase reporter assay verified the binding of the miR-424-5p and the 3’UTR of Bcl2. These results suggested that silencing miR-424-5p suppressed NP cell apoptosis by upregulating Bcl2. Therefore, miR-424-5p might be a novel target for IDD therapies.

中文翻译：

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miR-424-5p通过靶向髓核细胞中的Bcl2调节细胞凋亡和细胞增殖

摘要 miRNAs 在椎间盘退变（IDD）的发病机制中起重要作用。miR-424-5p 在人髓核 (NP) 中的作用和潜在机制尚不清楚。我们旨在探索 miR-424-5p 在 IDD 中的作用。Real-time PCR 检测 miR-424-5p 和 Bcl2 在 IDD 组织和特发性脊柱侧凸组织中的表达。我们的研究中使用了人类 NP 细胞。MTT和Hoechst凋亡实验分别用于检测NP细胞的增殖和凋亡。采用Western印迹法检测退行性NP细胞中Bcl-2、cleaved caspase-3、cleaved caspase-9、caspase-3和caspase-9的表达水平。应用荧光素酶报告基因测定来确认 miR-424-5p 和 Bcl2 之间的关系。我们的结果表明，在退行性 NP 细胞中，miR-424-5p 的表达增加，而 Bcl2 的表达减少。IDD组织中miR-425-5p的表达与Bcl2的表达呈负相关。使用抑制剂抑制 miR-424-5p 会增加 Bcl2 在 mRNA 和蛋白质水平的表达，并促进细胞活力并抑制细胞凋亡。此外，在 miR-424-5p 沉默的 NP 细胞中，cleaved caspase-3 和 cleaved caspase-9 的水平下调。有趣的是，我们发现沉默 miR-424-5p 会增加 p62 在 mRNA 和蛋白质水平的表达。最后，荧光素酶报告基因检测验证了 miR-424-5p 和 Bcl2 的 3'UTR 的结合。这些结果表明沉默 miR-424-5p 通过上调 Bcl2 抑制了 NP 细胞凋亡。所以，