ATRA, Neuroblastoma, Transcribed ultra-conserved regions, Differentiation
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.
Genetics | Medicine and Health Sciences
Watters KM, Bryan K, Foley NH, Meehan M, Stallings RL. Expressional alterations in functional ultra-conserved non-coding rnas in response to all-trans retinoic acid - induced differentiation in neuroblastoma cells. BMC Cancer. 2013;13:184
Untreated and ATRA-treated SK-N-BE cells. Cells were treated with ATRA (5μM final concentration) daily for 7 days. (A) Cells were probed using the neuronal marker βIII Tubulin (Abcam), and were incubated with the flourescein-conjugated goat anti-rabbit Alexa Flour 488 antibody (Invitrogen). (B) Western Blot for ßIII-Tubulin, MYCN and α-Tubulin (loading control) in untreated and ATRA-treated cells.
Additional file 2. Figure S2.tif (344 kB)
qPCR validation of tiling array results. Array results were validated for T-UC.324 and T-UC.300A by qPCR using gene-specific RT primers and custom-designed TaqMan Assays. Error bars represent standard deviation from the mean across at least two biological repeats.
Additional file 3. Table S1.xlsx (19 kB)
Correlation of intragenic T-UCR and host gene expression. T-UCRs are in four groups: exonic sense; exonic anti-sense; intronic sense; intronic anti-sense. Shaded cells denote significance or approaching significance.
Additional file 4. Figure S3.tif (320 kB)
qPCR validation of gene expression results. Gene expression microarray results were validated by qPCR for COL1A2 in SHSY5Y cells following knockdown of T-UC.300A. Figure shows results from array and from qPCR analysis. Error bars represent standard deviation from the mean across two biological repeats.