Investigation of the relationship between chromobox homolog 8 and nucleus pulposus cells degeneration in rat intervertebral disc.

Here, we aimed to investigate the expression of chromobox homolog 8 (CBX8) in nucleus pulposus (NP) cells from rat intervertebral disc (IVD) and its function in DNA damage and repair. NP cells were isolated from healthy rat IVD for immunohistochemistry staining. Small interfering RNA (siRNA) of CBX8 was applied for gene silencing, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine mRNA levels of CBX8, type II collagen, and proteoglycans. Cell proliferation and cell cycle were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and flow cytometry. Hydrogen peroxide (H2O2) was added to simulate DNA oxidative damage, and expression of CBX8 was examined using RT-PCR and Western blot. After five passages, mRNA levels of type II collagen and proteoglycans decreased but that of CBX8 increased. When CBX8 was silenced by siRNA, the expressions of CBX8, type II collagen and proteoglycans declined, and the cell growth was inhibited. Besides, cell cycle was slowed down as most cells were arrest in G0/G1 phase. Furthermore, CBX8 expression went up responding to DNA oxidative damage caused by H2O2. The data indicated that CBX8 plays important roles in cell proliferation and DNA damage. Cell proliferation and cell cycle were stimulated by CBX8, which may be associated with INK4A-ARF pathway. Moreover, CBX8 plays a role in DNA damage which made it a potential gene therapy target for treatment of disc degeneration.

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