Evaluation of epigenetic mechanisms regulating HOXD10, FGFR2 and ITIH5 gene expression in canine B-cell lymphoma: an in vitro approach.

Introduction. Despite B-cell Lymphoma (BCL) is the most common canine hematological cancer, there is still a lack of knowledge on molecular events contributing to its development and progression. Aim of this work was to investigate the epigenetic regulation of HOXD10, FGFR2 and ITIH5 genes, whose promoters were recently shown to be methylated, by genome-wide methylation profiling, in canine diffuse large BCL. To understand the role of methylation in silencing aforementioned genes, the CLBL-1 cell line was treated with two hypomethylating drugs (HDs). Owing to complexity of epigenetic mechanisms, histone deacetylase inhibitors (HDACis), alone or in combination with HDs, were also tested. Materials and methods. CLBL-1 cells were incubated with two HDs (azacytidine and decitabine), alone or in combination with HDACis (valproic acid, trichostatin and vorinostat), at concentrations corresponding to their IC50 and IC20 values (Alamar blue test), respectively. Then, gene methylation status and mRNA levels were measured using methyl sensitive PCR (MSP) and quantitative Real Time PCR (qPCR). Results. MSP showed an overall decrease of gene methylation status following the incubation with both HDs; meantime, qPCR highlighted a reversion of gene expression, confirming a methylation-dependent gene silencing mechanism. Interestingly, a higher pattern of gene re-expression was observed following the exposure to HDs combined with HDACis (and, mostly, with valproic acid). Moreover, HDACis alone increased the expression of FGFR2, demonstrating the important contribution of histone deacetylation, above methylation, in the regulation of this gene.