The long non-coding RNA BC200 (BCYRN1) is critical for cancer cell survival and proliferation

Abstract

Background: BC200 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.

Methods: Detailed 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 cytometry RESULTS: BC200 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.

Conclusions: BC200 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.

Keywords: Apoptosis; BC200; BCYRN1; Cancer; Cell cycle; Cell proliferation; Long noncoding RNA (lncRNA); Viability.

Fig. 1

Analysis of BC200 expression in normal human tissue, cancer cell lines and primary cells. (a) BC200 expression was assessed by RT-qPCR in a panel of eight human tissues. Absolute BC200 quantification data is plotted on the left y-axis whereas relative GAPDH expression is plotted on the right y-axis. Data represents the mean of three replicate measurements +/− standard deviation. (b) As in (a), BC200 and GAPDH expression were assessed in a panel of eight immortalized cell lines. (c) As in (a), BC200 and GAPDH expression were assessed in a panel of four primary and non-tumorigenic cell lines. (d) BC200 expression was assessed by northern blot in a panel of six cell lines relative to 5 fmoles of in-vitro transcribed BC200 (upper panel). Total RNA was visualized on a duplicate gel by staining with SYBR Gold (lower panel)

Fig. 2

BC200 expression is primarily cytoplasmic. (a) Relative BC200 expression in indicated subcellular fractions was assessed by RT-qPCR. 25 ng of RNA was used as a template from each fraction (equal input). Relative RNA abundance was corrected according to the portion of the total cellular RNA present in each fraction (abundance corrected). (b) As in (a) with primers specific to MALAT1 (c) As in (a) with primers specific to GRP94. (d) As in (a) with primers specific to GAPDH

Fig. 3

BC200 can be efficiently knocked down by siRNA and LNA GapmeR transfection. (a) Cells were transfected with a BC200 specific siRNA or GapmeR and harvested following 48-h. BC200 expression was assessed by RT-qPCR and normalized to the housekeeping gene GAPDH. (b) As in (a); however, absolute BC200 expression levels were assessed with a standard curve generated with the in-vitro transcribed RNA. (c) BC200 knock-down was confirmed in MCF-7 cells by northern blot with probes specific to a region in the 5′ end and 3′ end of the RNA. Total RNA was assessed by staining with SYBR Gold. The identity of the upper band in the middle panel is unknown

Fig. 4

BC200 knock-down by siRNA or LNA GapmeR reduces cell viability. (a) BC200 was knocked down in each of the indicated cell lines and viability was assessed over the course of 96 h by MTT assay. Non-targeting siRNA and GapmeR controls were transfected in an identical manner to control for transfection mediated toxicity. Viability was measured relative to untreated cells at each timepoint. Data represents the mean of six biological replicates +/− standard error. (b) MCF-7 cells were collected every eight hours following BC200 siRNA transfection and protein was isolated and analyzed by SDS/PAGE and western blot. To assess caspase cleavage, blots were probed with a Caspase-8 antibody as well as a tubulin antibody as loading control

Fig. 5

BC200 knock-down induces apoptosis. (a) BC200 was knocked down in the indicated cell lines by siRNA transfection. Cells were collected at the indicated time points and stained with Annexin V-Alexa Fluor 488 and propidium iodide to label apoptotic and dead cells. Data represents the fraction of unstained cells (lower left quadrant) at each timepoint measured in three biological replicates +/− standard error. (b) Dot plots of untreated, control siRNA and BC200 siRNA transfected MCF-7 cells at 72 h. Propidium iodide fluorescence is measured on the y-axis (FL3-H) and Annexin V-Alexa Fluor 488 is measured on the x-axis (FL1-H). (c) Apoptosis was assessed as in (a) following 96-h transfection with a BC200 targeting siRNA. (d) Apoptosis was assessed as in (a) following 96-h transfection with a BC200 targeting LNA GapmeR

Fig. 6

BC200 overexpression rescues the knock-down phenotype. (a) HeLa cells were transfected with expression vectors containing BC200 under control of the endogenous promoter (WT_BC200), BC200 under control of the U6 promoter (U6_BC200) as well as an siRNA resistant sequence mutant (U6_BC200 siMUT). BC200 was detected by denaturing TBE gel electrophoresis followed by northern blotting with DIG-labelled LNA probes targeting either the 3′ or 5′ ends of the RNA. Total RNA was detected in an identical gel run in parallel that was stained with SYBR gold. (b) HeLa cells were transfected with the indicated expression vectors for 24 h. 24 h post plasmid transfection cells were split an reverse transfected with BC200 siRNA or LNA GapmeR. Expression of endogenous and exogenous BC200 was assessed by qPCR with specific primers 48 h post transfection of the BC200 siRNA or LNA GapmeR. (c) Cell viability was measured by MTT assay 48 h post transfection of the BC200 siRNA or LNA GapmeR. Data represents the mean of 8 biological replicates +/− standard deviation. * indicates statistically significant deviation (p < 0.05) from Empty Vector transfected with BC200 siRNA. # indicates statistically significant deviation (p < 0.05) from empty vector transfected with BC200 LNA GapmeR. P-values were calculated by an unpaired two-tailed t-test. (d) The region of BC200 targeted by the LNA GapmeR and siRNA is shown in the context of the primary sequence and proposed secondary structure of BC200. The sequence scrambled in U6_BC200 siMUT (163-185) is shown alongside the wild-type sequence

Fig. 7

BC200 expression is reduced by confluence and serum deprivation. (a) Cells were plated at a concentration of 2 × 10⁵ (~50% confluent) or 5 × 10⁵ (100% confluent) and collected following 48-h culture. BC200 expression was measured by RT-qPCR and corrected to the housekeeping gene GAPDH. Data represents the mean of triplicate measurements +/− standard error. (b) Cells were plated at a concentration of 2 × 10⁵. Following 24-h media was changed to serum free DMEM. BC200 expression was assessed 24 h following serum deprivation by RT-qPCR as in (a)

Fig. 8

Cell cycle arrest reduces BC200 expression approximately 10-fold. (a) MCF-10A cells were treated with Lovastatin (40 μM), thymidine (2 mM), RO3306 (10 μM), nocodazole (0.1 μg/mL), or serum deprivation for 24 h to arrest cells in G1, S, G2, M and G1-phase respectively. BC200 expression was assessed by RT-qPCR and corrected to the housekeeping gene GAPDH. Data represents the mean of three replicates +/− standard error. Similar results were observed in HMEC cells (data not shown). (b) MCF-10A cells were arrested in the G1-phase by treatment with Lovastatin for 24 h. Cells were washed and released with mevalonic acid and collected every two hours to monitor BC200 and MYC expression and cell cycle phase. BC200 and MYC expression are represented on the left y-axis and the fraction of cells in each phase as determined by DNA content is represented by the right y-axis. (c) Histograms for selected time points demonstrate synchronous progression through the cell cycle following release from Lovastatin arrest. DNA content is monitored by propidium iodide fluorescence intensity on the x-axis (FL3-H)

Fig. 9

BC200 expression is regained upon growth factor reintroduction prior to exit from G1 phase. (a) MCF-10A cells were serum-deprived for 24 h followed by reintroduction of growth factors. Cells were collected every two hours to monitor BC200 and MYC expression by qPCR as well as cell cycle progression by flow cytometry. BC200 and MYC expression are represented on the left y-axis and the fraction of cells in each phase as determined by DNA content is represented by the right y-axis. (b) Histograms demonstrate the cell cycle profile of untreated and serum-deprived MCF-10A cells. (c) Histograms for selected time points demonstrate synchronous progression through the cell cycle following growth factor reintroduction

Fig. 10

Non-dividing cells are protected from viability loss due to BC200 knock-down. (a) MCF-10A cells were transfected with either control or BC200 targeting siRNA. Six-hours post-transfection media was changed to either complete media or media lacking growth factors (SF). BC200 expression was assessed 24-h post transfection by RT-qPCR. (b) Cells treated in the same manner as in (a) were employed for viability measurements by MTT assay over the course of 72-h post transfection. Data represents the mean of three biological replicates +/− standard error