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Validation of the Calca, Timp3, and Mmp2 Candidate Markers for Epigenetic / DNA Methylation Pattern Changes Associated with Cyclophosphamide-induced Chronic Cystitis
In-Seon Choi, PhD1, Abhishek Seth, MD2, Chester Koh, MD2.
1Children's Hospital Los Angeles / USC, Los Angeles, CA, USA, 2Texas Children's Hospital / Baylor, Houston, TX, USA.

Objective:
Epigenetics plays an important role in the regulation of inflammatory genes in chronic diseases such as asthma and chronic obstructive pulmonary disease (COPD). We recently described epigenetic / DNA methylation changes in select genes that occurred during non-cancer chronic bladder inflammation in an animal model. The purpose of this study was to determine if these epigenetic / DNA methylation changes were demonstrated during chronic bladder inflammation by pyrosequencing analysis.
Methods:
Chronic cystitis was induced in three groups of adult CD-1 male mice using multiple weight-based intraperitoneal cyclophosphamide (CYP) injections over a period of three months. Histopathologic and MethyLight assays were performed on chronic bladder inflammation specimens at multiple time points to monitor cystitis progression and DNA methylation changes in comparison to control specimens, respectively. Pyrosequencing analysis was performed to validate the candidate markers.
Results:
The most extensive edema and urothelial sloughing was seen at the 1-month time point by histopathological analysis. MethyLight analyses revealed statistically significant changes in DNA methylation associated with the Calca, Timp3, Mmp2, and Igf2r genes in the chronic bladder injury model, such as DNA hypomethylation of the Calca gene and DNA hypermethylation for the Timp3, Mmp2, and Igf2r genes, in comparison to control tissue. Pyrosequencing analysis for the Calca, Timp3, and Mmp2 loci during chronic cystitis showed that only the DNA methylation changes at the 1 and 2 month time points were similar with to those by MethyLight analysis for these 3 candidate genes.
Conclusions:
DNA methylation changes were noted in Calca, Timp3, and Mmp2 genes during chronic cystitis in a murine model by MethyLight and pyrosequencing analysis. Epigenetics / DNA methylation changes most likely play a role in the regulation of inflammatory bladder genes during chronic cystitis.


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