Abnormal stress promotes intervertebral disc degeneration through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2-mediated m6A modification in abnormal stress-induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Tension regulates the cartilage phenotypic expression of endplate chondrocytes through the α-catenin/actin skeleton/Hippo pathway.

The study aimed to investigate the regulatory mechanism of intracellular tension signaling in endplate chondrocytes and its impact on extracellular matrix synthesis. Human endplate chondrocytes were subjected to tension load using Flexcell FX-5000™, and changes in phenotype, morphology, and the expression of Hippo signaling pathway and α-Catenin were assessed through various techniques. Through the overexpression of YAP and inhibition of α-Catenin, the study clarified the intracellular tension signaling pathway and its regulation of extracellular matrix synthesis in endplate cartilage. In vitro-cultured human endplate chondrocytes significantly suppressed phenotype-related genes and proteins, accompanied by distinct changes in cytoskeleton morphology. Tension activation resulted in the substantial activation of the Hippo pathway, increased phosphorylation of YAP, and reduced nuclear translocation of YAP. YAP overexpression alleviated the inhibitory effect of tension on extracellular matrix synthesis in endplate chondrocytes. Tension also upregulated the expression of α-Catenin in endplate chondrocytes, which was attenuated by inhibiting α-Catenin expression, thereby reducing the impact of tension on cytoskeletal morphology and YAP nuclear translocation. Taken together, the α-Catenin/actin skeleton/Hippo-coupled network is a crucial signaling pathway for tension signaling in endplate chondrocytes, providing potential therapeutic targets for the treatment of endplate cartilage degeneration.

Codelivery of TGF-ß1 and anti-miR-141 by PLGA microspheres inhibits progression of intervertebral disc degeneration.

BACKGROUND: Cervical and lumbar pain is usually caused by degeneration of the nucleus pulposus (NP). As a powerful therapeutic strategy, tissue engineering can effectively restore the normal biological properties of the spinal unit. Previous studies suggested that poly(lactic-co-glycolic acid) (PLGA) microspheres are effective carriers of cells and biomolecules in NP tissue engineering. This study aims to explore the therapeutic effect of PLGA microspheres coloaded with transforming growth factor-ß1 (TGF-ß1) and anti-miR-141 on intervertebral disc degeneration (IDD). METHODS: PLGA microspheres were characterized by scanning electron microscopy, a laser particle size analyzer, and laser confocal microscopy. The in vitro release rate of biomolecules from the microspheres was analyzed by reversed-phase high-performance liquid chromatography and agarose gel electrophoresis. The rat NP cells (NPCs) treated with the solutions released from microspheres for different lengths of time were assigned to a control group (Ctrl), an empty PLGA microsphere group (Mock microsphere, MS), a TGF-ß1-loaded PLGA microsphere group (TMS), an anti-miR-141-loaded PLGA microsphere group (AMS), and an anti-miR-141 + TGF-ß1-loaded PLGA microsphere group (ATMS). The proliferation and apoptosis of NPCs were observed by alamar blue and flow cytometry. The gene and protein expression of cartilage markers COL2A1 and ACAN were observed by RT-qPCR and Western blot. The rat model of IDD was established by tail puncture. Rats were divided into a control group (Ctrl), a mock operation group (Mock), a TGF-ß1 microsphere group (TMS), an anti-miR-141 microsphere group (AMS), and an anti-miR-141 + TGF-ß1 microsphere group (ATMS). The degree of rat tail IDD was assessed in each group through magnetic resonance imaging (MRI), safranin O-fast green staining, immunohistochemistry, and Western blotting. RESULTS: PLGA microspheres were stably coloaded and could sustainably release TGF-ß1 and anti-miR-141. The results of in vitro cell experiments showed that the release solution of PLGA microspheres significantly enhanced the proliferation of NPCs without inducing their apoptosis and significantly upregulated cartilage markers in NPCs. The effect of microspheres was greater in the ATMS group than that in the TMS group and AMS group. In vivo experiments showed that IDD could be effectively inhibited and reversed by adding microspheres coloaded with TGF-ß1 and/or anti-miR-141, and the effect was greatest in the ATMS group. CONCLUSION: PLGA microspheres coloaded with TGF-ß1 and anti-miR-141 can reverse IDD by inhibiting the degeneration of NPCs.

N6-Methyladenosine-induced miR-143-3p promotes intervertebral disc degeneration by regulating SOX5.

Intervertebral disc degeneration is the basic cause of lumbocrural pain, which not only causes pain and but also serious economic burdens on patients. Increasingly more evidence has shown that tumor necrosis factor-α (TNF-α) is involved in the pathological process of intervertebral disc degeneration, but the specific molecular mechanism is still unclear. This study investigated the potential mechanism and function of methyltransferase-like 3 (METTL3)/miR-143-3p/SOX5 regulatory axis in nucleus pulposus cells under the action of TNF-α. Human nucleus pulposus cells were treated with TNF-α to construct an in vitro model of intervertebral disc degeneration. Flow cytometry, quantitative reverse-transcription PCR (RT-qPCR), Western blot (WB) and luciferase assays were used to identify the mechanism of action of miR-143-3p in the course of intervertebral disc degeneration in vitro and the downstream targeted regulatory molecules. The role of miR-143-3p in intervertebral disc degeneration was also validated by in vivo. RT-qPCR, WB, coimmunoprecipitation (Co-IP) and flow cytometry were used to verify the regulatory effect of METTL3 on miR-143-3p maturation. RT-qPCR and WB were adopted to detect differences in METTL3, miR-143-3p and SOX5 expression in human nucleus pulposus tissue. miR-143-3p in nucleus pulposus cells was involved in the regulation of extracellular matrix metabolism and apoptosis after TNF-α stimulation, and intervertebral disc degeneration was relieved by effectively regulating miR-143-3p expression. Subsequent experiments showed that the downstream direct target gene of miR-143-3p was SOX5 and that miR-143-3p negatively regulated the expression of SOX5. In addition, METTL3 promoted miR-143-3p maturation, and METTL3 and miR-143-3p were significantly upregulated in degenerative nucleus pulposus, an effect that was significantly negatively correlated with low SOX5 expression. In conclusion, TNF-α upregulates METTL3, METTL3 promotes miR-143-3p maturation, and miR-143-3p inhibits the transcriptional activity of SOX5 through targeted binding, thereby inducing intervertebral disc degeneration. The inhibition of METTL3 or miR-143-3p expression may be an effective way to treat intervertebral disc degeneration.

Circ_0022382 ameliorated intervertebral disc degeneration by regulating TGF-ß3 expression through sponge adsorption of miR-4726-5p.

Circular RNAs (circRNAs) participate in the progression of many diseases, but knowledge on the role of circRNAs in intervertebral disc degeneration (IDD) is limited. In this study, we discovered the characteristics of a new circRNA (circ_0022382) in human endplate chondrocytes. Currently, real-time quantitative polymerase chain reaction (RT-qPCR) showed that the relative expression level of circ_0022382 was significantly lower under intermittent cyclic tension stimulation than in the control group. circ_0022382, miR-4726-5p and Transforming growth factor 3 (TGF-ß3) were evaluated by RT-qPCR, Western Blot and immunofluorescence assay. Additionally, the role and mechanism of circ_0022382 in vivo were also consistent in the rat model. Furthermore, Intermittent cyclic mechanical tension can cause degeneration of endplate chondrocytes. The tension-sensitive circRNA_0022382 was decreased, and we found that circRNA_0022382 promoted morphology of endplate chondrocytes by sponge-binding miR-4726-5p down-regulation of target gene the TGF-ß3 expression, thereby alleviating IDD. In a rat model of acupuncture, intervertebral disc injection of circ_0022382 relieved the progression of IDD in vivo. In conclusion, the circ_0022382/miR-4726-5p/TGF-ß3 axis plays a key role in the anabolism and catabolism of the endplate chondrocyte extracellular matrix (ECM). It is suggested that circ_0022382 may provide a new approach for the prevention and treatment of IDD.