Nucleus Pulposus Extracellular Matrix Degradation Research Articles

CircRNA-CIDN mitigated compression loading-induced damage in human nucleus pulposus cells via miR-34a-5p/SIRT1 axis

BackgroundIntervertebral disc degeneration (IDD) is a major contributor to lower back pain, however, the molecular and pathogenetic mechanisms underlying IDD are poorly understood. As a high-risk factor for IDD, compression stress was reported to induce apoptosis of nucleus pulposus (NP) cells and extracellular matrix (ECM) degradation during IDD progression. Circular RNA (circRNA) is a class of endogenous non-coding RNA (ncRNA) and has been reported to function in several diseases. However, whether and how circRNA regulates compression-induced damage of NP cells remains vague. Here, we aimed to investigate the key role of circRNA in compression loading-induced IDD.MethodsWe analysed the circRNA expression of three samples from compression-treated NP cells and three control samples using circRNA microarray assays and further investigated the circRNA involved in compression-induced damage of NP cells (circRNA-CIDN). We investigated the effects of circRNA-CIDN on compression-induced cell apoptosis and NP ECM degradation in vitro and ex vivo. We observed that circRNA-CIDN bound to miRNAs as a miRNA sponge based on luciferase and RNA immunoprecipitation (RIP) assays.Findings:CircRNA-CIDN was significantly downregulated in compression-treated human NP cells, as validated by circRNA microarray and qRT-PCR analysis, and overexpressing circRNA-CIDN inhibited compression-induced apoptosis and NP ECM degradation. Further studies demonstrated that circRNA-CIDN served as a sponge for miR-34a-5p, an important miRNA that enhanced compression-induced damage of NP cells via repressing the silent mating type information regulation 2 homolog 1 (SIRT1). CircRNA-CIDN was also verified to contain IDD development in an ex vivo IDD model.InterpretationOur results revealed that circRNA-CIDN binding to miR-34a-5p played an important role in mitigating compression loading-induced nucleus pulposus cell damage via targeting SIRT1, providing a potential therapeutic strategy for IDD treatment.FundingNational Natural Science Foundation of China (81772391, 81974348), Fundamental Research Funds for the Central Universities (2017KFYXJJ248).

Inhibition of microRNA-34a prevents IL-1β-induced extracellular matrix degradation in nucleus pulposus by increasing GDF5 expression.

Accumulating evidence indicates that miRNAs, a class of small non-coding RNAs, are implicated in the pathogenesis of various diseases such as cancer and intervertebral disc degeneration. The aim of this study was to investigate the expression and the biological function of microRNA-34a in intervertebral disc degeneration. In this study, microRNA-34a expression was assessed in nucleus pulposus specimens and in IL-1β-stimulated nucleus pulposus cells by real-time polymerase chain reaction. microRNA-34a functions were investigated by using gain and loss of function experiments in nucleus pulposus cells and a dual luciferase reporter assay in 293T cells. microRNA-34a was dramatically up-regulated in degenerative nucleus pulposus tissues and in IL-1β-stimulated nucleus pulposus cells when compared with controls. Furthermore, growth differentiation factor 5 was identified as a target of microRNA-34a. Aberrant expression of microRNA-34a inhibited growth differentiation factor 5 expression by direct binding to its 3'-untranslated region. This inhibition was abolished by mutation of the microRNA-34a binding sites. In addition, microRNA-34a silencing reversed IL-1β-induced decrease in type II collagen and aggrecan expression in nucleus pulposus cells. This effect was substantially suppressed by growth differentiation factor 5 silencing. Our results suggested that microRNA-34a inhibition prevents IL-1β-induced extracellular matrix degradation in human nucleus pulposus by increasing growth differentiation factor 5 expression. microRNA-34a inhibition may be a novel molecular target for intervertebral disc degeneration treatment through the prevention of nucleus pulposus extracellular matrix degradation.

Role of IL-17 in nucleus pulposus cell proliferation and metabolism cultured in vitro.

To explore the role of cytokine, interleukin-17 (IL-17) in human degenerative disc disease. Through magnetic resonance imaging, human degenerative disc tissues were confirmed from the isolated nucleus pulposus cells, which were then cultured in vitro. The cells were cultured with and without different concentrations of IL-17. 2 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL and 20 ng/mL IL-17 concentrations were used for stimulation. After 72 hours, the inhibition rate of proliferation was measured by MTS method. For 48 and 96 hours, the nucleus pulposus cells were cultured with and without the appropriate IL-17 concentrations. The mRNA and protein expression levels of the matrix macromolecules and degrading tissue genes were measured by Real-time PCR and Western blot analysis. It was noted that nucleus pulposus cell proliferation was inhibited after culturing in vitro with IL-17 stimulation, and it was further observed that the inhibition effect was significantly stronger with 15 ng/mL IL-17 concentration. With the dosage of 15 ng/mL, IL-17 stimulation induced multiple cellular responses, such as the significant increase in mRNA expression for both aggrecan (ACAN) and type I collagen (COLLA1) genes (P<0.05), and the significant decrease in mRNA expression of both degrading tissue genes, MMP3 and TIMP3 (P<0.05). Western blot results also showed that the protein level of COL1A1 was significantly decreased (t=3.199, P=0.006), while the protein level of one peptidases (ADAMTS5) significantly increased (t=2.667, P=0.021). These findings suggest that IL-17 can inhibit proliferation and affect the metabolism of the cultured nucleus pulposus cells in vitro, and these findings could possibly contribute to the degenerative changes that occur in DDD through extracellular matrix synthesis inhibition, promoting nucleus pulposus extracellular matrix degradation and disrupting the metabolic balance.