Transfection of control siRNA and siRNA targeting gene of interest was performed using SilenceMag.
This article highlights the capacity of Magnetofection technology to efficiently silence gene expression into Kelly cells with siRNA using SilenceMag from OZ Biosciences.article reference: J Biol Chem. 2014 Aug 14. pii: jbc.M114.579391.
Shut Down of Achaete-Scute Homolog-1 Expression by Heterogeneous Nuclear Ribonucleoprotein (hnRNP)-A2/B1 in Hypoxia.
AbstractThe basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that the local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5`- and 3`-UTRs, while additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pull-down experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5`- and 3`-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.
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