Expressional Alterations in Functional Ultra-conserved Non-coding RNAs in Response to All-trans Retinoic Acid--induced Differentiation in Neuroblastoma Cells

Background: Ultra-conserved regions (UCRs) are segments of the genome (≥ 200 bp) that exhibit 100% DNA sequence conservation between human, mouse and rat. Transcribed UCRs (T-UCRs) have been shown to be differentially expressed in cancers versus normal tissue, indicating a possible role in carcinogenesis. All-trans-retinoic acid (ATRA) causes some neuroblastoma (NB) cell lines to undergo differentiation and leads to a significant decrease in the oncogenic transcription factor MYCN. Here, we examine the impact of ATRA treatment on T-UCR expression and investigate the biological significance of these changes.

Methods: We designed a custom tiling microarray to profile the expression of 481 T-UCRs in sense and anti-sense orientation (962 potential transcripts) in untreated and ATRA-treated neuroblastoma cell lines (SH-SY5Y, SK-N-BE, LAN-5). Following identification of significantly differentially expressed T-UCRs, we carried out siRNA knockdown and gene expression microarray analysis to investigate putative functional roles for selected T-UCRs.

Results: Following ATRA-induced differentiation, 32 T-UCRs were differentially expressed (16 up-regulated, 16 down-regulated) across all three cell lines. Further insight into the possible role of T-UC.300A, an independent transcript whose expression is down-regulated following ATRA was achieved by siRNA knockdown, resulting in the decreased viability and invasiveness of ATRA-responsive cell lines. Gene expression microarray analysis following knockdown of T-UC.300A revealed a number of genes whose expression was altered by changing T-UC.300A levels and that might play a role in the increased proliferation and invasion of NB cells prior to ATRA-treatment.

Conclusions: Our results indicate that significant numbers of T-UCRs have altered expression levels in response to ATRA. While the precise roles that T-UCRs might play in cancer or in normal development are largely unknown and an important area for future study, our findings strongly indicate that the function of non-coding RNA T-UC.300A is connected with proliferation, invasion and the inhibition of differentiation of neuroblastoma cell lines prior to ATRA treatment.