All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumorinitiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factorb/bone morphogenetic protein, Wnt/b-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n ¼ 82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n ¼ 8) and significantly increased with tumor grade progression (Po0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways.
MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity.
BackgroundMicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir.Methodology/Principal FindingsWe employed a proteomics technique called “stable isotope labelling by amino acids in cell culture” (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation.Conclusions/SignificanceTo the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs.
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