Department of Orthopaedics, Huashan Hospital, Fudan University, Shanghai, China, 200040.
Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China, 200032.
Exp Mol Med. 2018 Aug 6;50(8):1-12. doi: 10.1038/s12276-018-0125-y.
This study was carried out to explore the roles of circular RNAs (circRNAs) in nucleus pulposus (NP) tissues in intervertebral disc degeneration (IDD). Differentially expressed circRNAs in IDD and normal NP tissues were identified based on the results of microarray analysis. Bioinformatics techniques were employed to predict the direct interactions of selected circRNAs, microRNAs (miR), and mRNAs. CircRNA_104670 was selected as the target circRNA due to its large multiplier expression in IDD tissues. After luciferase reporter and EGFP/RFP reporter assays, we confirmed that circRNA_104670 directly bound to miR-17-3p, while MMP-2 was the direct target of miR-17-3p. The receiver-operating characteristic (ROC) curve showed that circRNA_104670 and miR-17-3p had good diagnostic significance for IDD (AUC = 0.96; AUC = 0.91). A significant correlation was detected between the Pfirrmann grade and expression of circRNA_104670 (r = 0.63; p = 0.00) and miR-17-3p (r = -0.62; p = 0.00). Flow-cytometric analysis and the MTT assay showed that interfering with circRNA_104670 using small interfering RNA (siRNA) inhibited NP cell apoptosis (p < 0.01), and this inhibition was reduced by interfering with miR-17-3p. Interfering with circRNA_104670 suppressed MMP-2 expression and increased extracellular matrix (ECM) formation, which were also reduced by interfering with miR-17-3p. Finally, an MRI evaluation showed that circRNA_104670 inhibition mice had a lower IDD grade compared with control mice (p < 0.01), whereas circRNA_104670 and miRNA-17-3p inhibition mice had a higher IDD grade compared with circRNA_104670 inhibition mice (p < 0.05). CircRNA_104670 is highly expressed in the NP tissues of IDD and acts as a ceRNA during NP degradation.
本研究旨在探讨环状 RNA（circRNA）在椎间盘退变（IDD）中髓核组织中的作用。基于微阵列分析的结果，鉴定了 IDD 和正常 NP 组织中差异表达的 circRNAs。利用生物信息学技术预测选定的 circRNA、microRNA（miR）和 mRNAs 的直接相互作用。由于 circRNA_104670 在 IDD 组织中的表达倍数较大，因此选择其作为靶 circRNA。通过荧光素酶报告基因和 EGFP/RFP 报告基因检测，我们证实 circRNA_104670 可直接与 miR-17-3p 结合，而 MMP-2 是 miR-17-3p 的直接靶标。受试者工作特征（ROC）曲线表明，circRNA_104670 和 miR-17-3p 对 IDD 具有良好的诊断意义（AUC = 0.96；AUC = 0.91）。circRNA_104670 的表达与 Pfirrmann 分级呈显著正相关（r = 0.63；p = 0.00），与 miR-17-3p 的表达呈显著负相关（r = -0.62；p = 0.00）。流式细胞术分析和 MTT 检测结果显示，用小干扰 RNA（siRNA）干扰 circRNA_104670 可抑制 NP 细胞凋亡（p < 0.01），而干扰 miR-17-3p 则降低了这种抑制作用。干扰 circRNA_104670 可抑制 MMP-2 表达并增加细胞外基质（ECM）形成，干扰 miR-17-3p 也可降低这一作用。最后，MRI 评估结果显示，与对照组相比，circRNA_104670 抑制组的 IDD 分级较低（p < 0.01），而 circRNA_104670 和 miR-17-3p 抑制组的 IDD 分级较 circRNA_104670 抑制组更高（p < 0.05）。circRNA_104670 在 IDD 的 NP 组织中高表达，并在 NP 降解过程中作为 ceRNA 发挥作用。
