Aberrant expression of microRNA in gliomas: Molecular mechanisms, functional consequences and clinical significance

Coordinator:		Dr. rer. nat. Marietta Wolter
Institution:		Neuropathologie, Universitätsklinik Düsseldorf
Homepage:		http://www.uniklinik-duesseldorf.de/neuropathologie

The aim of this project is the identification as well as the molecular and functional characterization of aberrantly expressed microRNAs in malignant gliomas. The first milestone was an expression profiling of 365 human microRNAs (miRNAs) using TaqMan Low Density Arrays and real-time RT-PCR in a series of 125 primary gliomas, 10 non-neoplastic brain tissue samples, and 35 glioma cell lines. Expression profiling of four cell lines treated with demethylating agents and histon deacetylase inhibitors vs. untreated control cells revealed 46 miRNAs with increased expression (Fold change ? 2,0)  in at least two of the four investigated cell lines. The three miRNAs selected for further analyses miR-330 (19q13.32), miR-132 (17p13.3), miR-126 (9q34.4) exhibited decreased expression in the gliomas investigated compared to the expression in non-neoplastic brain tissue samples. Using sequencing of bisulfite-modified DNA, hypermethylation of CpG-sites located in the 5´-genomic regions of the miRNAs was identified in the tumor tissues compared to non-neoplastic brain tissues. In the case of miR-132 and miR-126 modifications of histons H3 and H4 seemed to be involved in the regulation of transcription. The over-expression of the three miRNAs in glioma cells resulted in a significant increase in apoptotic activity. Putative target genes of these miRNA have been identified and validated. Using an expression analysis of 16 miRNAs located on chromosomal region 10q we identified four miRNAs (miR-107, miR-146b, miR-346, miR-1287) exhibiting a significantly decreased expression in high grade astrocytomas compared to normal brain tissues. Hypermethylation of CpG sites in 5´-located regulatory sequences seemed to be involved in the decreased expression of the miR-346 and miR-146b in the astrocytic tumors. The over-expression of miR-107, miR-146b, and miR-346 in glioma cells resulted in a significant increase in apoptotic activity. Putative targets have been identified and validated by Luciferase reporter gene assays for all four miRNAs. Thereby, we contribute to the overall research goal of the NGNGplus IG Hirntumore by providing comprehensive information on the role of miRNA in glioma pathogenesis, elucidating the relationships between miRNA aberrations and glioma-associated genetic, epigenetic and translational changes, and assessing the possible clinical significance of miRNA expression in gliomas.