Abstract
microRNAs are an abundant class of small non-coding RNAs that control gene expression post-transcriptionally. Importantly, microRNA activity participates in the regulation of cellular processes and is a potentially valuable source of biomarkers in the diagnosis and prognosis of human diseases. Here we introduce miQPCR, an innovative method to quantify microRNAs expression by using Real-Time PCR. miQPCR exploits T4 RNA ligase activities to extend uniformly microRNAs’ 3′-ends by addition of a linker-adapter. The adapter is then used as ‘anchor’ to prime cDNA synthesis and throughout qPCR to amplify specifically target amplicons. miQPCR is an open, adaptable and cost-effective procedure, which offers the following advantages; i) universal elongation and reverse transcription of all microRNAs; ii) Tm-adjustment of microRNA-specific primers; iii) high sensitivity and specificity in discriminating among closely related sequences and; iv) suitable for the analysis of cellular and cell-free circulating microRNAs. Analysis of cellular and cell-free circulating microRNAs secreted by rat primary hepatocytes stimulated with cytokines and growth factors identifies for the first time a widespread modulation of both microRNAs expression and secretion. Altogether, our findings suggest that the pleiotropic activity of humoral factors on microRNAs may extensively affect liver function in response to injury and regeneration.
Highlights
MicroRNAs are short, non-coding RNAs that control gene expression at the post-transcriptional level1. miRNAs are transcribed within long primary transcripts that are processed via two successive RNase III-like enzyme mediated-cleavage steps
We identified that FGF2, FGF4 and INF-β, down-regulated the expression of several miRNAs in the cultured hepatocytes, while IL-6, INF-β and TGF-β 1 are able to modulate the quantity of exosomal-miRNAs secreted by the primary hepatocytes into the culture medium
Results miQPCR workflow, miRNA elongation and reverse transcription. miRNA expression profiling is challenging as mature miRNAs are; i) short single stranded RNAs (22–24 nts); ii) their CG content varies between 33% of hsa-miR-144 and 89% of hsa-miR-4665-3p resulting in a wide range of Tms; iii) miRNAs only represent a small fraction of the cellular RNA; iv) miRNA target sequence is contained in its precursors; and v) miRNAs are redundant and exist in families where individual members can differ by just a single nucleotide[27]
Summary
MicroRNAs (miRNAs) are short, non-coding RNAs (ncRNAs) that control gene expression at the post-transcriptional level1. miRNAs are transcribed within long primary transcripts that are processed via two successive RNase III-like enzyme mediated-cleavage steps. Two different populations of cell-free circulating miRNAs have been identified; one included in exosomes and one associated to the Argonaute proteins[18]. To the best of our knowledge, this is the first study showing that cytokines and growth factors are able to modulate both the expression and the secretion of miRNAs in cultured primary hepatocytes. We identified that FGF2, FGF4 and INF-β , down-regulated the expression of several miRNAs in the cultured hepatocytes, while IL-6, INF-β and TGF-β 1 are able to modulate the quantity of exosomal-miRNAs secreted by the primary hepatocytes into the culture medium. Our data indicate that the pleiotropic effect of cytokines and growth factors on miRNAs expression and secretion might have an extensive effect on liver function whilst the liver is recovering from different insults or during chronic and acute liver diseases
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