Abstract
MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene.
Highlights
Neuroblastoma (NB),1 a neoplasm that arises from embryonic neural crest tissue, is the second-most common solid pediatric tumor [1]
To produce the chimeric transcripts, the human MYCN 3Ј-untranslated regions (UTRs) was subcloned into the pBBB vector in a unique BglII site within the 3Ј-UTR of the rabbit -globin gene to generate pBBN (Fig. 1A). pBBN was co-transfected with pSV2neo into National Institutes of Health (NIH) 3T3 cells and neomycinresistant colonies were selected
RNA Stabilization by the embryonic lethal abnormal vision-like (ELAV) RNA-binding Protein HuD—To test our hypothesis that MYCN mRNA stabilization is the functional consequence of interactions between HuD and cis-acting elements within the MYCN 3Ј-UTR, we overexpressed HuD in NIH 3T3 cells stably transfected with pBBN (3T3 BBN)
Summary
Construction of Recombinant Plasmids—Genomic DNA was isolated from W-N NB cells and used in a PCR to amplify the 3Ј-untranslated region of the MYCN gene. In the first series of constructs, pBB(F1–F8)B, the MYCN 3Ј-UTR fragments were cloned into the unique BglII site of pBBB between the -globin coding region and the -globin 3Ј-UTR. The forward primers used to generate the series II fragments contained a BglII site and the reverse primer a SacI site to facilitate cloning into the BglII/SacI sites immediately downstream of the -globin coding region of pBBB. Stable Transfections—NIH 3T3 cells were co-transfected with 2 g of pBBB, pBBN, or chimeric reporter constructs containing MYCN 3ЈUTR fragments (pBB(F1–F8)B or pBB(F1–F8)) and 0.2 g of pSV2neo (ATCC, Rockville, MD) with 10 l of SuperFect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Real-time RT-PCR Analysis of MYCN Expression—Total RNA was isolated from W-N cells treated with sense or antisense oligodeoxynucleotides. The expression of MYCN was normalized to 2-microglobulin message levels, and the data are expressed as the CT value relative to 2-microglobulin (2-microglobulin CT Ϫ MYCN CT)
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