Abstract
BackgroundBC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.MethodsDetailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometryResultsBC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.ConclusionsBC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.
Highlights
BC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types
We show that BC200 expression is greatly reduced in senescent and arrested cells and is elevated upon resumption of the cell cycle, suggesting the possibility of a distinct role for this long non-coding RNA outside of the nervous system
Evaluation of BC200 expression in normal human tissue and tumour derived cell lines As much of the previously published expression analyses of BC200 relied upon Northern blotting and in-situ hybridization methodologies [3, 7, 8], we sought to assess BC200 expression by performing direct quantification of RNA copy number by real-time quantitative PCR (RT-qPCR)
Summary
BC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. Despite a lack of sequence homology, primate-specific BC200 demonstrates a similar expression pattern as the mouse BC1 RNA with high neuronal levels and dendritic localization [1, 3]. In addition to a similar expression pattern, both BC1 and BC200 have been found to globally repress translation in both in vitro translation assays and when co-transfected with reporter mRNAs into HeLa cells [12,13,14,15]. In light of these results, BC200 is postulated to play a role in localized translational control in neuronal cells; specific mRNA targets of BC200 remain to be elucidated
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