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Epigenetic and genetic alterations of microRNAs (miRNAs) are frequently seen in cancer, and are responsible for the deregulation of differentiation and proliferation programs. The levels of transcription of miRNAs have been directly linked to epigenetic silencing, i.e. hypermethylation of CpG islands in tumors. Osteosarcoma is the most common primary malignant bone tumor and shows complex genomic aberrations. Using Illumina's Infinium technology we have profiled global promoter methylation of the EuroBoNeT osteosarcoma cell line panel (eurobonet.eu), in addition to a small panel of normal samples (osteoblast primary cultures and normal bone). A list of miRNAs that could be associated to a promoter CpG island was generated by identifying miRNAs that are encoded within transcripts of protein-coding host genes, or within 10 kb from a CpG island. Using an integrative approach, we have analyzed miRNA expression patterns together with DNA methylation data. Interestingly, hypermethylation of the upstream promoter seems to strongly suppress miRNA expression, and a high inverse correlation was observed between the degree of DNA methylation and expression of 17 miRNAs. The identified miRNAs were associated with hypermethylation in a high proportion of the osteosarcoma cell lines and hypomethylation in normal samples, suggesting that their transcription is regulated by promoter methylation, and that their silencing may play a role in osteosarcoma biology. To study this further, a subset of the cell lines was treated with a DNA demethylation agent. The relative expression levels of 8 miRNAs were found to increase upon treatment, as determined by quantitative RT-PCR. Thus, the transcription of these miRNAs seems to be regulated by epigenetic mechanisms. The methylation status of CpG islands associated with the most interesting miRNAs was then analyzed in more detail using methylation-specific PCR and direct bisulphite sequencing. We are currently expanding these investigations to a large tumor set where we will use quantitative methylation-specific PCR as a tool to determine the methylation status. Based on our findings, methylation of CpG islands seems to play a crucial role in controlling miRNA expression in osteosarcomas. This reveals new possibilities for activation of tumor-suppressing miRNAs by epigenetic treatment of osteosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 197. doi:1538-7445.AM2012-197