Abstract
Introduction
The intervertebral disc (IVD) cell distribution depends on the developmental stage of the human disc. DNA methylation has been shown to be essential to mammalian development, and aberrant methylation patterns are a common feature of many kinds of age-related diseases. We hypothesize that the methylation status of the promoter region of the genomic DNA regulates gene expression of IVD cells.
Materials and Methods
IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures with Institutional Review Board approval. The tissues were transported to the laboratory, minced, and immediately underwent cell isolation by sequential enzyme digestion. The digested cells were centrifuged and seeded in culture dishes, supplemented with complete medium (DMEM/F12) containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotic. The demethylation agent 5-azacytidine (5-aza-C) was used to inhibit DNA methylation in IVD cells. The cells were then plated and treated with 5-aza-C followed by RNA isolation and real time PCR for gene analysis.
Results
We investigated important cytokines (IL-1b, IL-6), anabolic genes (Aggrecan, Collagen type II), apoptotic (Bax), and cell cycle (p16, cyclin D2) genes following 5-aza-C. In the presence of 5-azaC treatment, IL-1b, IL-6, Bax, p16, and cyclin D2 expression were upregulated approximately twofold or more. The anabolic gene aggrecan was downregulated whereas no effect on collagen type II expression.
Conclusion
The experimentally induced loss of DNA methylation results in the increase expression of apoptotic, catabolic, and inflammatory genes. Furthermore, anabolic genes aggrecan significantly downregulated in 5-azacytidine treated samples, thus confirming a cause-and-effect relationship between DNA demethylation and transcription of these genes in IVD cells. DNA demethylation has been previously shown to inhibit cell proliferation and induce apoptotic cell death in different cell types. Our results suggest that demethylation of IVD cells may be one of the influential causes for IVD degeneration. Further studies are required to confirm our findings.
None declared