Because methylation analysis of CL tissue specimens was originally performed

without the use of microdissection, we assumed that a “dilution effect” by nonparenchymal cells (mainly fibrocytes and fibroblasts) might conceal hypermethylation of hepatocytes in these samples. However, even improved analysis employing microdissected CL samples of the same tissue specimens failed to confirm promoter hypermethylation as the cause of AKAP12 down-regulation in CL and DN. In search of posttranscriptional mechanisms for AKAP12 find more down-regulation we detected an alternative regulatory mechanism in CL and DN by miR-183 and miR-186. Both of these miRNAs are up-regulated in the precancerous stages where promoter hypermethylation is absent; and, via a direct interaction with the AKAP12 transcript, both miRNAs can regulate mRNA levels to various degrees, with miR-186 demonstrating a strong ability to regulate endogenous transcript levels. Regarding the observed genetic and epigenetic alterations in HCC, this represents an interesting interplay between different epigenetic regulatory mechanisms in the course of human hepatocarcinogenesis. A connection between epigenome and miRNome and alteration in the balance of this complicated network as a possible mechanism leading to cancer has been described recently.24 Additional mechanisms may also account for AKAP12 down-regulation.

In CL, it could be shown that a histone deacetylase inhibitor influences SSeCKS expression.25 Apart from aberrant patterns of histone modification, involvement of chromatin modifications in the expression of the AKAP12α isoform was recently shown Ceritinib price by its re-expression after treatment of mouse fibroblasts with a histone deacetylase

inhibitor.26 Different models this website support the hypothesis that CpG island methylation may follow histone modification to stably lock silenced genes.27 It is therefore conceivable that the observed de novo DNA methylation of the AKAP12α promoter in HCCs may be also triggered by histone modifications which are already present in CL. In summary, the data presented here demonstrate that the tumor suppressor AKAP12 is down-regulated during hepatocarcinogenesis in a stepwise manner: early in cirrhosis and in premalignant lesions, and late in HCC dedifferentiation. We could identify different epigenetic mechanisms responsible for this stepwise down-regulation. In CL and DN, down-regulation of AKAP12 is at least partly caused by interaction of two specific miRNAs, whereas in HCC genetic loss and to a significant extent hypermethylation of the AKAP12α promoter are responsible for AKAP12 reduction. We thank Peter Waas, Anna-Lisa Lackner, and Otto Zelezny (Division of Epigenomics and Cancer Risk Factor, German Cancer Research Center), and Eva Eiteneuer and John Moyers (Institute of Pathology, University of Heidelberg) for their excellent technical assistance. Additional Supporting Information may be found in the online version of this article.