DNA methylation is, besides other mechanisms like histone modifications, part of the epigenetic gene silencing machinery which leads to inactivation of certain tumor suppressor genes (TSG) in cancer. Numerous TSGs have already been identified which are frequently methylated in lung cancer and transcriptionally silenced. While DNA methylation is reversible by DNA methyltransferase inhibitors like 5-aza-2′-deoxycytidine (Aza-dC), histone deacetylation can be inhibited by histone deacetylase inhibitors like trichostatin A (TSA). Overall, there is a synergistic effect in gene re-expression using both drugs. Recently, it has been observed that also microRNA (miRNA) encoding genes may be targets for methylation in cancer. miRNAs are short, noncoding RNAs which act as post-transcriptional regulators of gene expression and therefore are involved in regulation of various biological processes. So far, about 900 human miRNAs have been identified and it has been suggested that altered expression of certain miRNAs is involved in tumorigenesis. However, information about regulation of expression of miRNA encoding genes is limited so far. To investigate the role of epigenetic miRNA gene silencing in lung cancer, we performed a genome-wide microarray based screen for differentially expressed miRNAs in non-small cell lung cancer A549 cells before and after treatment with Aza-dC or a combination of Aza-dC and TSA. Total RNA from untreated and drug treated cells was size fractionated, Cy3/Cy5 labeled and hybridized to dual-channel microarrays (designed by LC Sciences according to miRBase 12.0 covering 853 miRNAs). Microarray data were normalized and t-statistics were performed to identify miRNAs differentially expressed before and after drug treatment. 28 miRNAs were identified whose expression was up-regulated in Aza-dC treated A549 cells and 32 miRNAs whose expression was up-regulated in Aza-dC/TSA treated cells. Overall, expression of 41 miRNAs was up-regulated after Aza-dC or Aza-dC/TSA treatment of A549 cells. 21 out of these 41 (52%) miRNA genes are associated with a CpG island. Some of these 21 miRNAs (e.g. hsa-let-7a, hsa-miR-27b) were already found to be down-regulated in lung cancer tissue, however, the mechanism of silencing was unknown. The genes encoding for these miRNAs will be further analysed for methylation in additional lung cancer cell lines and in primary lung cancer samples. In conclusion, we identified several miRNA encoding genes whose expression seems to be regulated by epigenetic mechanisms in lung cancer. Overall, our results suggest that epigenetic silencing might be an important mechanism for inactivation of certain miRNA encoding genes in lung cancer.

This work was supported by the Vienna Science and Technology Fund (WWTF, project number LS07-019).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 177.

©2010 American Association for Cancer Research
2010