Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration.
Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration.
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miR-133a-5p/FBXO6轴在椎间盘退变调控中的作用
DOI：
10.1016/j.jot.2021.05.004
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发表时间：
2021-07
影响因子：
6.6
通讯作者：
Zheng ZM
中科院分区：
文献类型：
--
作者：
Du XF;Cui HT;Pan HH;Long J;Cui HW;Chen SL;Wang JR;Li ZM;Liu H;Huang YC;Wang H;Zheng ZM
腰痛是世界范围内致残的主要原因，而椎间盘退变（IVDD）相关疾病已被认为是主要原因之一。然而，其潜在的机制尚未被完全理解。本研究的目的是探讨miR-133a-5p/FBXO6轴在IVDD调控中的作用。RT-qPCR、WB和IHC检测FBXO6在人IVD组织中的表达。用IL-1β处理髓核（NP）细胞，建立IVDD细胞模型。使用特定的sirna实现FBXO6的沉默。采用CCK-8法、流式细胞术、TUNEL法、RT-qPCR及WB法评价FBXO6在IVDD过程中的作用及机制。使用在线工具、GSE数据集和RT-qPCR搜索靶向FBXO6的候选mirna。FBXO6和miR-133a-5p之间的直接结合位点通过双荧光素酶实验进一步验证。通过RT-qPCR、WB和救援实验，鉴定miR-133a-5p对aggrecan、collagenⅡ、MMP3、ADAMTS5、IL-6、COX2表达的调控功能。此外，利用慢病毒shRNA、WB和RT-qPCR研究NF-κB通路在miR-133a-5p调控中的作用。结果显示，FBXO6主要在IVD的NP组织中表达，且FBXO6的表达随IVDD过程及IL-1β刺激而降低。FBXO6的沉默导致聚集蛋白和胶原Ⅱ的表达降低，MMP3、ADAMTS5、IL-6、COX2的表达增加，进一步诱导NP细胞的变性。生物信息学分析表明，miR-133a-5p是靶向FBXO6的候选miRNA。miR-133a-5p在IVDD组织中上调，显著抑制FBXO6的表达。抑制miR-133a-5p可改善体外FBXO6沉默诱导的IVDD加速。此外，IL-1β通过NF-κB通路调节NP细胞中miR-133a-5p/FBXO6轴的表达。IL-1β上调miR-133a-5p通过海绵FBXO6加重椎间盘退变。抑制miR-133a-5p表达或挽救FBXO6表达可能是治疗IVDD的有希望的策略。本研究提示miR-133a-5p/FBXO6轴能够调控NP细胞的增殖、凋亡、细胞外基质的合成和降解，为IVDD的治疗提供了一个有希望的治疗靶点和策略。
Low back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD. RT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1β to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR. Results showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1β stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1β regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells. miR-133a-5p was upregulated by IL-1β to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD. This study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.
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