Abstract 2766: Genome-wide miRNA methylation analyses in non-small cell lung cancer patients
Authors: Heller Gerwin , Altenberger Corinna , Topakian Thais , Ziegler Barbara , Lang György , End-Pfützenreuter Adelheid , Steiner Irene , Zehetmayer Sonja , Döme Balazs , Klepetko Walter , Posch Martin , Zielinski Christoph C. , Zöchbauer-Müller Sabine 
Published: July 2016

Abstract
Deregulated DNA methylation (referred to as methylation) leading to transcriptional inactivation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). Besides protein encoding genes also microRNA (miRNA) encoding genes were found to be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. Thus, we investigated genome-wide methylation of miRNA genes in primary tumors (TU) and corresponding non-malignant lung tissue samples (NL) of 50 NSCLC patients using methylated DNA immunoprecipitation followed by custom designed tiling microarray analyses (MeDIP-chip). Differentially methylated miRNA genes between TU and NL samples were identified using paired t-statistics with permutation adjusted p-values for step down multiple testing. Using this approach, 201 differentially methylated probes between TU and NL samples were found. These probes were annotated resulting in the identification of 39 tumor-specifically methylated miRNA genes. 79% of them are associated with a 5′ CpG island. While some of these miRNA genes were already known to be methylated in NSCLCs (e.g. miR-9-3, miR-124) methylation of the vast majority of them was unknown so far.
We selected 6 miRNA genes (miR-10b, miR-1179, miR-137, miR-572, miR-3150b and miR-129-2) for gene-specific methylation analyses in TU and corresponding NL samples of 108 NSCLC patients and observed statistically significant tumor-specific methylation of these miRNA genes which confirmed our MeDIP-chip data (p&lt;0.0001, respectively). Using miRWalk2.0 software, we searched for predicted targets of the 6 miRNAs and identified several oncogenic/cell proliferation promoting factors. Examples of them are TFAP2C and HOXA1 (predicted miR-10b targets), HMGB3, CCNE1 and FGF11 (predicted miR-1179 targets), HOXD11 and ONECUT1 (predicted miR-572 targets). By analysing RNA-seq data of &gt; 1.000 NSCLC patients from The Cancer Genome Atlas database, we found that many of the predicted miRNA targets are upregulated in TU samples (e.g. CCNE1, HMGB3, HOXD11, TFAP2A, ZIC2). Based on these findings, we investigated if miR-1179 indeed targets CCNE1 in vitro. Thus, we transfected miR-1179 mimics into CCNE1 expressing NSCLC cell lines (HTB182 and NCI-H1650). Using RT-PCR, we found downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls. In addition, we observed downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls by Western blot analyses. Additional functional analyses of miR-1179 targets as well as of other miRNA targets are ongoing.
In conclusion, we identified a large number of tumor-specifically methylated miRNA genes in NSCLC patients and found that some of these miRNAs target certain oncogenes in NSCLC cells. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs.
Citation Format: Gerwin Heller, Corinna Altenberger, Thais Topakian, Barbara Ziegler, György Lang, Adelheid End-Pfützenreuter, Irene Steiner, Sonja Zehetmayer, Balazs Döme, Walter Klepetko, Martin Posch, Christoph C. Zielinski, Sabine Zöchbauer-Müller. Genome-wide miRNA methylation analyses in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2766.