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Exploring the Mechanism by which circPTK2 Affects Apoptosis of Human Nucleus Pulposus Cells Based on the WNT/β-Catenin Pathway
Vol 37, Issue 8, 2023
Abstract
Background: Intervertebral disc degeneration-related disease (IVDD) involves the gradual deterioration of the structure and function of intervertebral discs. Understanding the pathogenesis of IVDD is crucial for developing effective prevention and treatment strategies. In recent years, the abnormal acceleration of apoptosis in nucleus pulposus cells (NPCs) has been identified as a major factor in the progression of IVDD. Reversing this pathological process is considered a potential breakthrough in the diagnosis and treatment of IVDD. Studies have indicated an association between hsa_circ_0008305 (circPTK2) and the Wingless/Integrated (WNT)/β-catenin pathway, which regulates cell growth. This suggests that circPTK2 may be linked to the progression of IVDD through its ability to modulate the biological behavior of NPCs. Our goal is to investigate how circPTK2 influences interleukin (IL)-1β-induced apoptosis of human nucleus pulposus cells (NPCs) through the WNT/β-catenin pathway, and to identify early biological markers for IVDD and provide clues for therapeutic targets. Methods: NPCs cells were cultured and treated with IL-1β to establish a model group of IVDD. Meanwhile, untreated NPC cells were established as a control group. Next, we performed qRT-PCR quantitative analysis on the expression of circPTK2 and WNT/β-catenin in the cells. In order to further study the role of circPTK2 in IVDD model cells, we performed interference experiments. We introduced the circPTK2 small interfering vector into IVDD model cells to form the si-circPTK2 panel. At the same time, we introduced the circPTK2-negative sequence as a control to form the si-NC group. To assess cell viability and apoptosis, we used Cell Counting Kit-8 (CCK-8) reagent and flow cytometry, respectively. In addition, we also used western blotting technology and Enzyme-Linked Immunosorbent Assay (ELISA) to measure the contents of IL-6, IL-8, IL-10 and tumor necrosis factor-α (TNF-α) in the cell supernatant to evaluate the inflammatory response in the cells. Results: Both circPTK2 and WNT/β-catenin presented higher levels in the model group than in the control group (p < 0.05). Inhibiting circPTK2 caused enhanced proliferative capacity of IVDD model cells and a reduced apoptosis rate (p < 0.05). The apoptotic proteins (Bax and cleaved Caspase3) in the si-circPTK2 group were significantly higher than in the control group but lower than in the model and si-NC groups (p < 0.05), while the anti-apoptotic protein Bcl-1 was higher than in the model and si-NC groups (p < 0.05). The test results of inflammatory cytokines (ICs) identified lower pro-ICs (IL-6, IL-8 and TNF-α), but higher anti-IC (IL-10) in the si-circPTK2 group compared with the model and si-NC groups (p < 0.05). WNT/β-catenin suppression reverses the inhibitory effect of circPTK2 on apoptosis in nucleus pulposus cells (p < 0.05). Conclusion: circPTK2 is highly expressed in IVDD. After inhibiting circPTK2, the proliferation of NPCs is increased, and the apoptosis and inflammatory reactions are decreased, which may be realized through the WNT/β-catenin pathway.
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Medical Genetics, University of Torino Medical School, Italy

Department of Biomedical, Surgical and Dental Sciences, University of Milan, Italy