Argonaute-miRNA complexes reveal concerted action in disease related pathways of a human, glioblastoma cell line

Glioblastoma is the most common primary malignant brain tumor occurring in the central nervous system and is characterized by rapid proliferation, genetic aberrations and poor response to treatment. The classical genetic alterations in glioblastoma target pathways governing cellular proliferation, cellular survival, invasion and angiogenesis. In this study, the regulation of glioblastoma-relevant pathways by small non-coding RNAs was analyzed by identification of Argonaute protein-associated microRNAs and mRNAs. We utilized a highly controlled method termed PAR-CLIP-Array by biochemical isolation of cross-linked ribonucleoprotein complexes with monoclonal antibodies specific for individual Argonaute proteins followed by microarray detection. We demonstrate here that the different Argonaute proteins bind different subsets of miRNAs and mRNAs in a glioblastoma cell line model. By extending our study generating miRNA-mRNA network interaction models we could further show, that different Argonaute proteins act in concert to regulate glioblastoma-relevant pathways. We therefore provide novel insights into glioblastoma regulation by microRNAs. Co-immunoprecipitation in the glioblastoma cell line model SNB19 of three different Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4'-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by an antibody isotype control and empty bead controls. Argonaute-associated miRNAs/mRNAs and uspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. The empty bead controls were not hybridized to the microarrays, but only used for correction of unspecific RNA binding to the bead matrix during Western Blot and RT-PCR validation. The total RNA of SNB19 cells was hybridized to microarrays for comparison with Ago-associated RNAs. These experiments were performed in triplicates.