Abstract
Introduction
Intervertebral disc (IVD) degeneration is one of the most important aging health problems and a leading cause of disability. However, little is known about pathophysiology of this condition. This study investigates the role of epigenetic factor in intervertebral disc gene regulation.
Material and Methods
IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures. The collected tissues were minced and immediately used for cell isolation by sequential enzyme digestion. The demethylation agent 5-azacytidine (5-AzaC) was used to inhibit DNA methylation in IVD cells. The cells were then analyzed for change in DNA methylation using Methyl-Profiler PCR array. We used Methyl-Profiler PCR array containing developmental and cell cycle regulatory genes. The Methyl-Profiler PCR arrays were performed as per manufacturer's instructions. Briefly, the cells were treated with and without 5-azaC for 4 days, and genomic DNA were isolated. Then the equal amounts of genomic DNA were overnight digested with methyl-specific restriction digestion enzyme, nonspecific restriction enzyme, a combination of methyl specific and non-specific, and a control mock digestion. Following digestion, real time PCR was performed using biorad-MyiQ cycler. The data was analyzed according to manufacturer's instructions.
Results
Methyl-Profiler PCR array showed novel genes hypo-methylated following 5-azacytidine treatment. We found that around 6 genes' promoters differentially methylated after experimental demethylation in IVD cells. The genes differentially methylated are CCND2, CDH1, HIC1, MGMT, PRDM2 and TP73. Real time PCR showed these genes were differentially regulated relative to control.
Conclusion
There are reports that these differentially methylated genes have a role in various age related degenerative diseases. We speculate that these genes may also play an important role in intervertebral disc degeneration. It would be interesting to investigate the expression of these genes and their methylation pattern in aging human degenerative IVD. In summary, our results suggest that demethylation of IVD cells may be one of the factors involved in IVD degeneration. Further studies are required to confirm our findings and to better understand the effects of aberrant DNA methylation on IVD biology and disease pathophysiology.