MicroRNA-497 increases apoptosis in MYCN amplified neuroblastoma cells by targeting the key cell cycle regulator WEE1

Analysis of miR-497 expression levels in 143 primary diagnostic neuroblastoma samples (Additional file 1: Table S1) revealed significantly lower expression of miR-497 (based on median expression) in patients with known higher risk prognostic factors including MYCN amplification (MNA) and INSS Stage 4 disease (Figure 1A). Although miR-497 was found not to be independent of other known risk factors (Additional file 2: Table S2), higher expression (> first quartile) of miR-497 was significantly associated with both improved event free survival (EFS 5 year 65% vs 16%) and overall survival (OS 5 year 84% vs 21%), indicating a potential tumor suppressor role in neuroblastoma (Figure 1B).

To further investigate a potential tumor suppressor role of miR-497 in neuroblastoma, we observed the effects of miR-497 over-expression on neuroblastoma cell viability, by transiently transfecting mature miR-497 mimics into MNA Kelly and CHP-212 cell lines and non-MNA SK-N-AS cell line. MiR-497 expression was significantly up-regulated following transfection in all cell lines (Additional file 3: Figure S1). Ectopic expression of miR-497 resulted in significantly decreased cell viability in the MNA Kelly and CHP-212 cell lines at 96 hr relative to negative controls, while lowered cell viability observed in SK-N-AS was not statistically significant (Figure 2A).

Consistent with these results, a significant increase in apoptosis activity was observed in MNA Kelly and CHP-212 cells following miR-497 transfection, as determined by Annexin V/PI staining and FACs analysis at 96 hr following miR-497 transfection (Figure 2B). No change in the rate of apoptotic cell death was detected for SK-N-AS (Figure 2B). Representative scatter plots for Kelly and CHP-212 are illustrated (Figure 2D). A significant increase in caspase 3/7 activation was observed in MNA Kelly and CHP-212 cells when compared to negative controls. Consistent with both cell viability and Annexin V assays, no change in caspase activation was observed in non-MNA SK-N-AS cells (Figure 2C). We also note that Kelly and CHP-212 cells became rounded and detached following miR-497 over-expression, consistent with apoptosis, whereas SK-N-AS cells remained attached to the dish (Figure 2E). We conclude that miR-497 decreases cell viability in MNA neuroblastoma Kelly and CHP-212 cells through an increase in apoptotic rates. Consistent with the above results, cell cycle analysis of MNA Kelly cells revealed a significant increase in cell number in the G₀/G₁ phase of the cell cycle following over-expression of miR-497 when compared to negative controls. A corresponding significant decrease in the number of cells in the G₂/M of the cell cycle was observed, following over-expression of miR-497 when compared to negative controls. These results further support the apoptotic phenotype following miR-497 over-expression (Additional file 4: Figure S4A). Representative cell cycle analysis plots of MNA Kelly cells are illustrated (Additional file 4: Figure S4C). MiR-497 targets the 3′UTR of WEE1 in neuroblastoma cells. Through In silico analysis (using the algorithms TargetScan, miRDB and miRanda), we examined all computationally predicted target genes of miR-497. WEE1 was a computationally predicted target of interest given our observed phenotypic effect of decreased cell viability and increased apoptotic rates following miR-497 over-expression, and WEE1’s documented role as a negative regulator of CDC2 mediated apoptosis [29]. WEE1 has two conserved 7-mer seed matches with miR-497 in its 3′UTR (Figure 3A).

Ectopic expression of miR-497 mimics in Kelly, CHP-212 and SK-N-AS cells resulted in knockdown of WEE1 protein but not mRNA, indicating that the miRNA had a potential inhibitory effect on translation (Figure 3B,C). Densitometry analysis shows significant decrease in WEE1 protein levels following miR-497 over-expression when compared to negative controls (Figure 3D). To determine if miR-497 directly targets the 3′ UTR of WEE1, luciferase reporter plasmids were constructed containing a 450 bp segment of the WEE1 3′ UTR with either the wild type or a mutated miR-497 seed site. As miR-497 has two potential binding sites in the 3′UTR of WEE1, mutant reporter constructs were made that had either single mutated sites or both sites mutated (Additional file 5: Figure S2). Co-transfection of the reporter construct containing the wild-type binding sequence with mature miR-497 mimics resulted in a statistically significant reduction in luciferase activity in Kelly cells (Figure 3E). Luciferase activity was also significantly reduced in both reporter constructs with only one of the two potential miR-497 binding sites mutated. This effect was abrogated by a double mutated target sequence, thereby confirming that WEE1 is directly targeted by miR-497 (Figure 3E).

Analysis of WEE1 expression levels in 88 primary diagnostic neuroblastoma samples revealed a significant association of high WEE1 expression with poor EFS and OS. Further analysis of this data set revealed no significant difference in the median expression of WEE1 in tumors with/without MNA but a significant difference for WEE1 median expression levels between tumors from INSS Stage 4 versus Stage 1,2,3 and 4 s disease (Figure 4A).

To test the hypothesis that miR-497 targeting of WEE1 might contribute, in part, to the biological effects of miR-497, we performed siRNA mediated inhibition of WEE1. WEE1 was significantly reduced at both mRNA and protein levels following siWEE1 transfection of Kelly, CHP212 and SK-N-AS cells (Additional file 6: Figure S3A and B). siWEE1 resulted in significantly decreased cell viability in Kelly, CHP-212 and SK-N-AS cell lines at both 72 hr and 96 hr time points relative to negative controls (Figure 4B). Given that siRNA mediated inhibition of WEE1, a now validated target of miR-497, had a similar phenotypic effect on our neuroblastoma cell lines, Annexin V/PI assays were performed using siWEE1.

Following WEE1 inhibition, significantly increased levels of apoptosis (Annexin V⁺) were observed relative to negative controls at 96 hr for both Kelly and CHP-212. However, in SK-N-AS cells, there was not a significant increase in apoptotic cell numbers (Figure 4C). siRNA mediated inhibition of WEE1 in Kelly and CHP-212 cells resulted in the cells becoming rounded and detached, similar to the morphological changes observed following miR-497 over-expression. SK-N-AS cells, however, retained their morphology and remained attached to the dish, consistent with results produced by miR-497 ectopic over-expression. Taken together, our results indicate that WEE1 knockdown mediated by either miR-497 or siRNA in SK-N-AS results in a reduction in cell proliferation without causing increased apoptosis (Figure 4D). Consistent with the above results, cell cycle analysis of MNA Kelly cells revealed a significant increase in cell number in the G₀/G₁ phase of the cell cycle following WEE1 inhibition when compared to negative controls. A corresponding significant decrease in the number of cells in the G₂/M of the cell cycle following WEE1 inhibition was observed, when compared to negative controls. These results further support the apoptotic phenotype following WEE1 inhibition (Additional file 4: Figure S4B). Representative cell cycle analysis plots of MNA Kelly cells are illustrated (Additional file 4: Figure S4D).

In order to elucidate how miR-497 may contribute to improved patient survival, we assessed the effect of miR-497 over-expression and siRNA mediated knockdown of WEE1 on apoptotic rates in Kelly, CHP-212 and SK-N-AS cell lines in response to CDDP. Following transfection with miR-497 mimics and treatment with 5ug/ml CDDP, a significant increase in apoptotic cells (Annexin V⁺) was detected in Kelly and CHP-212 (Figure 5A) relative to negative controls. These cell lines also displayed increased apoptosis in response to siRNA mediated WEE1 inhibition and treatment with 5ug/ml CDDP (Figure 5B). SK-N-AS exhibited a significant increase in apoptosis in response to siRNA mediated WEE1 inhibition and treatment with 5ug/ml CDDP, but not in response to miR-497 and CDDP (Figure 5A,B), compared to negative controls. Representative scatter plots for Kelly are illustrated (Figure 5C,D).

Primary neuroblastoma tumor samples (n=143) were obtained from the Children’s Oncology Group (COG), Philadelphia, USA (n=112) or from Our Lady’s Children’s Hospital, Dublin, Ireland (n=31) (Additional file 1: Table S1). Research was approved by the Research Ethics Committees of the Royal College of Surgeons and Our Lady’s Children’s Hospital, Dublin. Detailed miRNA expression profiling of this cohort of patients is described previously [9]. An independent data set of 88 primary neuroblastoma tumors was also used as part of the analysis for this study. This data is readily available using the web based R2 microarray analysis and visualization platform from the Academic Medical Center (AMC), Amsterdam (http://​hgserver1.​amc.​nl/​cgi-bin/​r2/​main.​cgi).

Neuroblastoma cell lines including Kelly and CHP-212 (MYCN amplified) and SK-N-AS (non-MYCN amplified) were purchased from the European Collection of Animal Cells. All lines were validated by short tandem repeat sequence genotyping and for presence of previously published genomic imbalances using array comparative genomic hybridisation (aCGH). Cell culture media was supplemented with 10% FBS and 1% Pen/Strep.

Viability of cells was measured by MTS-formazan reduction using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) at 24 hr, 48 hr, 72 hr, and 96 hr post transfection. Absorbance was measured at 490 nm using a Synergy Multi-Mode Plate Reader (Boitek, Winooski, VT, USA). Apoptosis levels were demonstrated by Annexin-V staining and propidium iodide (PI) exclusion using the FITC Annexin-V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Cells were acquired using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA) and analysed using BD FACSDiva 4.0 Software. Caspase 3/7 activity was evaluated using the Caspase-Glo® 3/7 Assay (Promega) and luminescence recorded using a Synergy Multi-Mode Plate Reader (Boitek).

Total RNA was extracted from cell lines using miRNeasy Mini Kits (Qiagen, Valencia, CA, USA). Reverse transcription was performed using total RNA with primers specific for miR-497 or RNU48 control and TaqMan microRNA reverse transcription kit (Applied Biosystems Life Technologes, Carlsbad, California, USA). For gene expression analysis, reverse transcription was performed using High-Capacity reverse transcription kits (Applied Biosystems). Specific TaqMan assays (Applied Biosystems) for WEE1 and miR-497 were employed for expression analysis on the 7900 HT Fast Realtime System (Applied Biosystems). MiRNA and gene expression was normalised using the endogenous controls RNU48 and 18S respectively and relative quantities determined by the delta CT method [39].

Total protein was analysed by western blotting using primary antibodies anti-WEE1 (B11) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A), followed by anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA USA) and anti-mouse alpha-tubulin loading control (Abcam,Cambridge, MA USA).

Cell cycle progression and proliferation was monitored using the Cell Cycle Assay Kit - Green Fluorometric at 48 hr post transfection (Abcam Cambridge, MA USA). Cells were acquired using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA) and analysed using Weasel 3.1 Software