Abstract
MicroRNAs are positive and negative regulators of eukaryotic gene expression that modulate transcript abundance by specific binding to sequence motifs located prevalently in the 3' untranslated regions of target messenger RNAs (mRNA). Interferon-alpha-2a (IFNα) induces a large set of protein coding genes mediating antiproliferative and antiviral responses. Here we use a global microarray-based microRNA detection platform to identify genes that are induced by IFNα in hepatoma- or melanoma-derived human tumor cell lines. Despite the enormous differences in expression levels between these models, we were able to identify microRNAs that are upregulated by IFNα in both lines suggesting the possibility that interferon-regulated microRNAs are involved in the transcriptional repression of mRNA relevant to cytokine responses.
Highlights
The gene expression patterns of tumor-derived cell lines differ greatly, as do their responses to antiproliferative effects of interferons (IFNs)
In contrast to secondary response genes (SRGs), all Primary response genes (PRGs) studied to date contain bona fide interferon response elements in the promoter region, which are required for binding of the interferon-stimulated gene factor 3 (ISGF3) complex and for janus kinase/signal transducer of transcription (JAK/STAT)pathway-mediated signaling
Melanoma cells (ME-15) were cultured in RPMI 1640 with L-Glutamine supplemented with non-essential amino acids and sodium pyruvate (1 mM) and hepatoma (HuH7) cells were cultured in DMEM + GlutaMAX
Summary
A hallmark of the therapeutic activity of type I interferons is the induction of antiproliferative activity mediated by the upregulation of several hundred response genes with pleiotropic functions (1). Some recent reports showed that interferon beta (IFNβ) stimulation can boost microRNA levels in cell culture together with inhibition of viral replication (9) At this point it is an open question whether this induction is IFNβ specific or a shared feature of all type I interferons. HuH7 is commonly used as a model for testing antiviral effects of IFN in the HCV replicon system In both models efficient responses to IFNα have been shown at the functional and transcriptional level. We have chosen a DNA-microarray-based technology (Illumina) for the multi-parallel expression analysis of all known human microRNAs (http://microrna.sanger.ac.uk/; Release 10.0: August 2007) This method allowed us to process total RNA as template, allowing the possibility of mRNA gene expression profiling in further experiments. PCR amplification is performed with fluorescently labeled primers, which allows quantitative signal detection by conventional confocal laser scanning
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