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circSNTB2 and CUL4A Induces Dysfunction of Nucleus Pulposus Cells by Competitively Binding miR-665.
Biochemical Genetics 2023 July 29
Circular RNA (circRNA) plays important roles in lumbar degenerative diseases. This study aimed to investigate the role of circSNTB2 in regulating the development of lumbar disc herniation (LDH) in vitro and in vivo. The abnormally expressed circSNTB2 in intervertebral disc degeneration (IDD) through bioinformatics analysis was identified, and verified in nucleus pulposus (NP) tissues of patients with LDH. NP cells were treated with TNF-α to mimic the LDH microenvironment. RT-qPCR was applied to determine levels of mRNA and microRNA (miRNA) in clinical samples and cells. We performed CCK-8, EdU, TUNEL and flow cytometric apoptosis assays to evaluate the proliferation and apoptosis of NP cells. The predicted the miRNAs and downstream target genes were verified with the help of luciferase reporter gene and RNA pull-down experiments. Finally, we established an LDH rat model to further verify the role of circSNTB2 in vivo. circSNTB2 was significantly up-regulated in the NP tissues of LDH group and TNF-α -treated NP cells. miR-665 binds to circSNTB2 and cullin 4A (CUL4A) is the downstream target gene of miR-665. Knockdown of circSNTB2 promoted NP cells proliferation and inhibited apoptosis, which was reversed by down-regulation of miR-665. In addition, up-regulated CUL4A reversed the effects of over-expressed miR-665 on proliferation and apoptosis of NP cells. Meanwhile, results of in vivo experiments demonstrated that knocking down circSNTB2 alleviated LDH-induced thermo-mechanical pain and NP injury. In summary, circSNTB2 regulates the proliferation and apoptosis of NP by mediating miR-665 regulation of CUL4A, which provides a reliable idea for targeted therapy of LDH. Graphical abstract of how circSNTB2 promotes LDH progression. A schematic model of circSNTB2/miR-665/CUL4A signaling pathway in NP cells. CircSNTB2 competitively binds to miR-665, resulting in upregulation of CUL4A.
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