Session Type: Abstract Submissions (ACR)
DNA methylation is one of the best-characterized epigenetic modifications that have been implicated in numerous biologic and pathologic processes. It is initiated by DNA methyltransferases (DNMT) 3a and 3b and maintained in subsequent cellular generations by the maintenance enzyme DNMT1. Despite its role in long-term gene silencing, DNA methylation is now believed to be a dynamic process as active DNA demethylation has been observed in certain experimental systems. Thus, during cellular division a competition between DNMT1 and DNA demethylase determine the maintenance or the reversal of DNA methylation in the daughter cells. The molecular identity of DNA demethylase remains elusive but its activity can be measured by functional assay. DNMT1 expression is upregulated in SSc microvascular endothelial cells (MVEC) and fibroblasts (FB) in association with persistence of DNA methylation of the NCpG islands in the promoter region of key underexpressed genes (i.e. Fli1 and NOS3). Thus we thought in this study to examine the activity of DNA demethylase in SSc and control cells and the role of microRNA (miRNA) in the regulation of DNA demethylase activities.
MVEC and FB were isolated from involved SSc skin and control subjects. DNMT1 expression levels were determined by qRT-PCR and by western blot analysis. DNA demethylase activity was measured in nuclear extracts using the EPI Quik™ DNA Demethylase Activity/Inhibition Assay Kit. Small RNA molecules including miRNA were isolated from SSc and control MVEC and FB using PureLink miRNA isolation kit. The effects of miRNA on DNA demethylase activity was examined in SSc and control cells by transfecting the cells with SSc and control miRNA.
The following results were observed in this study:
- DNMT1 expression levels were significantly upregulated in SSc cells (mean 3.2 and 2.8 folds in MVEC and FB respectively vs control cells, mean 3 cell lines each).
- DNA demethylase activity was significantly reduced in SSc cells (42% and 51 % in MVEC and FB respectively vs control cells, mean 3 cell lines each)
- The knockdown of DNMT1 using DNMT1 specific siRNA did not affect demethylase activity.
- Transfection of SSc cells with control miRNA results in decrease expression of DNMT1 and increase activity of DNA demethylase, while the transfection of control cells with SSc miRNA resulted in upregulation of DNMT1 and reduced DNA demethylase activity.
- SSc MVEC and FB transfection with control miRNA normalized abnormal gene expression profile.
Conclusion: This study demonstrates upregulation of DNMT1 and diminished activities of DNA demethylase in SSc MVEC and FB and that DNA demethylase activity is regulated by miRNA. The characterization of the molecular mechanisms that target both DNA methylation and demethylation is essential for understanding the emergence and persistence of the pathologic phenotype exhibited by SSc cells.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/decrease-activity-of-dna-demethylase-in-ssc-fibroblast-and-microvascular-endothelial-cells-a-possible-mechanism-for-persistence-of-ssc-phenotype/