Abstract
Abstract Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behaviour, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in NB tumors is highly correlated with poor survival. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid to reduce minimal residual disease, and exposure of a number of NB cell lines to the compound all-trans-retinoic acid (ATRA) induces neural differentiation along with the down-regulation of MYCN. Thus, identification of targets regulated by either ATRA or MYCN is of potential interest for the development of novel therapeutics. Ultra-conserved regions (UCRs) are sequences ≥200bp in length that are 100% conserved between human, mouse and rat genomes. In NB, transcribed UCRs (T-UCRs) exhibit specific profiles in MYCN-amplified (MNA) compared to non-MNA tumors (Mestdagh et al., Oncogene 2010 29:3583), and in high risk versus low risk tumors (Scaruffi et al., BMC Cancer 2009 9;441). Here we identify T-UCRs that are responsive to ATRA as well as T-UCRs that are directly or indirectly regulated by MYCN, and evaluate the biological significance of selected T-UCRs in functional studies of NB cell lines. We designed a custom tiling microarray to profile the expression of 481 UCRs in sense and antisense orientation (962 potential T-UCRs) in three ATRA-sensitive NB cell lines, and in SHEP-21N cells which contain a MYCN trans-gene controlled by a tetracycline responsive repressor element. Thirty-two T-UCRs were differentially expressed following ATRA treatment across all three ATRA sensitive cell lines (p<0.05), while analysis of doxycyclin-treated SHEP-21N cells (to deplete MYCN levels) indicated that 42 T-UCRs were altered by changing MYCN levels. In addition we identified a small number of T-UCRs, including T-UC.396 (intergenic) and T-UC.324 (antisense to host gene), whose expression was altered in both systems. These T-UCRs may represent independent ncRNAs directly or indirectly regulated by MYCN. Functionality of T-UC.300A and T-UC.324 was assessed by siRNA knockdown. While knockdown of T-UC.324 had no effect on cell viability or invasion, depletion of T-UC.300A resulted in a significant decrease in both cell viability and invasion. Our results indicate that significant numbers of T-UCRs have altered expression levels in response to changing MYCN levels and ATRA-induced differentiation. While the precise roles that these ncRNAs might play in cancer or in normal development are largely unknown, we have identified a T-UCR, T-UC.300A, whose down-regulation results in the decreased viability and invasiveness of ATRA-responsive cell lines and may play a functional role in ATRA-induced differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 202. doi:1538-7445.AM2012-202
Published Version
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