Background
Several pediatric cancers feature amplification at the chromosomal region 2p24 including alveolar rhabdomyosarcoma (ARMS), neuroblastoma (NB), medulloblastoma, Wilms’ tumor, and retinoblastoma [
1‐
3]. The minimum common region of amplification at 2p24 in rhabdomyosarcoma (RMS) and NB has been found to consistently include the oncogene
MYCN and amplification of
MYCN is used clinically as a prognostic marker in NB [
3‐
7]. Amplification or overexpression of
MYCN leads to dysregulation of proliferation, differentiation and the cell cycle in NB [
8] and contributes to cell growth in ARMS [
9]. MYCN is also capable of positive auto-regulation as well as auto-suppression in NB, potentially fine-tuning MYCN levels [
10‐
12]. In ARMS,
MYCN transcription is driven by PAX3-FOXO1, the protein product of a fusion between the
PAX3 and
FOXO1 genes that has prognostic significance in these tumors [
9,
13]. RMS and NB are a major cause of cancer related death in children with a five-year survival rate of around 50% for high-risk NB cases, including those with
MYCN amplification, and 40% for
PAX3-FOXO1 positive RMS cases [
13,
14].
MYCN opposite-strand (
MYCNOS, N-CYM, MYCN-AS1, NYCM, CYMN) is produced by antisense transcription across exon 1 and intron 1 of
MYCN that has been shown to be highly expressed in
MYCN-amplified NB and small cell lung cancer [
15,
16]. Two alternative transcripts denoted
MYCNOS-01 and
MYCNOS-02 (Additional file
1: Figure S1A) are fully sequence-verified [
15]. There is an emerging body of evidence for roles of
MYCNOS-02 through an encoded protein (NCYM) that promotes NB tumorigenesis, in particular via its regulation of
MYCN expression, and also its role as a long non-coding RNA (lncRNA) [
17‐
22]. NCYM has been shown to mediate expression of MYCN protein by both direct interaction and also indirectly via inhibition of GSK3β, leading to decreased MYCN phosphorylation and a concomitant increase in MYCN protein stability [
17]. This was associated with increased tumor growth and metastasis [
17]. The same study also concluded that MYCN positively drives the promoter of
MYCNOS-02 in an E-box-dependent manner [
17]. As well as increasing MYCN protein, NCYM has been found to increase MYCN cleavage to produce the anti-apoptotic protein Myc-nick in NB [
22]. NCYM has also been shown to promote aggressiveness in NB by increasing
OCT4 expression via its stabilization of MYCN [
20].
LncRNAs are commonly defined as transcripts of over 200 nucleotides in length that in general do not code for a protein [
23].
MYCNOS-02 lncRNA is able to regulate the usage of two
MYCN promoters and therefore expression of different
MYCN transcripts via interaction with binding partners such as G3BP1. This in turn results in expression of different isoforms of the MYCN protein [
18].
MYCNOS-02 lncRNA has also been found to recruit CTCF to the
MYCN promoter to increase recruitment of activating chromatin marks and thus increase
MYCN expression [
19]. This positive regulation of
MYCN suppressed differentiation and increased growth, invasion and metastasis in NB [
19]. Additionally, a recent study has shown
MYCNOS-02 lncRNA can interact with the RNA-binding protein NonO to indirectly increase
MYCN transcript levels post-transcriptionally [
21]. Overall, these studies show that both the
MYCNOS-02 encoded protein and lncRNA play a role in growth, invasion and metastasis of NB cells [
17,
19,
20].
Unlike
MYCNOS-02, a functional role for the
MYCNOS-01 transcript has not yet been investigated, despite original annotation of its sequence being consistent with a lncRNA [
15]. In this study we therefore investigated the role of
MYCNOS-01 as a lncRNA in RMS and NB. We demonstrate that
MYCNOS-01 post-transcriptionally regulates MYCN protein levels without affecting
MYCN mRNA levels, whilst MYCN regulates
MYCNOS-01 transcription. We show that silencing of
MYCNOS-01 in RMS and NB cell lines with
MYCN amplification reduces cell viability, similar to the effects of
MYCN reduction. Thus, we conclude that regulation of MYCN by
MYCNOS-01 contributes to the reduction in cell growth in RMS and NB cell lines after
MYCNOS-01 silencing.
Methods
Translation and Kozak sequence prediction tools
Cell culture
Human ARMS cell line RMS-01 was available directly from the authors [
26] and the RH30 cell line was a gift from Peter Houghton (St Jude Children’s Research Hospital, Memphis, Tennessee). The human NB cell lines KELLY and SY5Y were obtained from ECACC (cat. No. 92110411) and ATCC (cat. No. CRL-2266) respectively. RMS-01 and RH30 were cultured in DMEM (Thermo Fisher Scientific, MA, USA) and KELLY and SY5Y were cultured in RPMI-1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% Foetal Bovine Serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin. The
MYCN overexpressing and matched empty vector expressing RH30 lines were generated as previously described in Tonelli et al., (2012) [
9] and cultured in DMEM supplemented with 400 μg/ml geneticin. Cells were maintained at 37 °C and 5% CO
2. Data from short tandem repeat testing of the cell lines using the GenePrint 10 system (Promega, WI, USA) were compared with records for these cell lines in a repository database or our own archival records. This was consistent with the origin of these cell lines.
Analyses of expression profiling data
Data uploaded to R2 Genomics Analysis and Visualisation Platform (
http://r2.amc.nl) were used for analyses. These included 101 RMS samples (ITCC) [
6], a set of 19 RMS cell lines (Versteeg) [
27], 88 NB samples (Versteeg) and 24 NB cell lines (Versteeg) that had been previously profiled using the Affymetrix GeneChip with the HGU133 Plus2 array. Probe sets could distinguish
MYCNOS-01 and
MYCNOS-02 transcripts: probe set 216188_at detects
MYCNOS-01, set 207028_at detects
MYCNOS-02 and set 209757_s_at was used to detect
MYCN.
qRT-PCR
MYCNOS-01 and
MYCNOS-02 expression data was available from primary sample biopsies from RMS patients. Samples and details of RNA extraction were previously described [
6,
28] with appropriate approvals for investigation. RNA was isolated from cell lines using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Cell line cDNA was synthesised using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, CA, USA) and patient sample cDNA was synthesised using SuperScript II reverse transcriptase (Invitrogen, CA, USA) following the manufacturers’ protocol. Samples were run for qRT-PCR on the ViiA™ 7 Real-Time PCR System (Applied Biosystems, CA, USA). The following Taqman
® probe and primer sets were used:
MYCNOS-01 Hs01032821_m1,
MYCNOS-02 Hs01040745_m1,
MYCN Hs_00232074_m1. Human
ACTB (Beta Actin) endogenous control (Applied Biosystems, CA, USA) was used to normalise gene expression. Each sample was run in triplicate. Analysis of
MYCN expression and copy number in patient samples is described in [
6].
siRNA transfection
Oligonucleotides for specific silencing of
MYCNOS-01, MYCNOS-02 and
MYCN were transfected into cells using Lipofectamine RNAimax (Invitrogen, CA, USA) according to the manufacturer’s instructions. All siRNAs were obtained from GE Dharmacon (CO, USA). The sequence from 5′ to 3′ for the three siRNAs against
MYCNOS-01 were as follows: siMYCNOS-01 1 GGGACAAGAGCACAGUUUCUU, siMYCNOS-01 2 GGUAAGUUAAGGUACAGCCUU, siMYCNOS-01 3 GGAGUAUUUGUUUAGUGCUUU. The sequences for the three siRNAs against
MYCNOS-02 were GAAAGAAGGGUAGUCCGAAUU for siMYCNOS-02 1, GACCGAUGCUUCUAACCCAUU for siMYCNOS-02 2, CCGCUUUGACUGCGUGUUGUU for siMYCNOS-02 3. For knockdown of MYCN a pool of three siRNAs was used with sequences GAAGAAAUCGACGUGGUCA, CCAAGGCUGUCACCACAUU, AAUUGAACACGCUCGGACU, as previously described [
9]. The control siRNA used was the ON-TARGETplus non-targeting control pool (GE Dharmacon, CO, USA). Samples were analyzed by qRT-PCR, Western blot, flow cytometry or phenotypic assays at time-points indicated in the relevant figures.
Western blotting
Protein lysates were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) and their concentration measured by the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Protein samples were resolved by SDS-PAGE and transferred onto PVDF membranes. Blots were incubated with the following primary antibodies: MYCN SC-791 (1:200, Santa Cruz, TX, USA), PARP 9542 (1:1000, Cell Signaling Technology, MA, USA), Phospho-C-Myc (Thr58/Ser62) 04–217 (1:4000, Merck Millipore, MA, USA), GAPDH MAB374 (1:10000, Merck Millipore, MA, USA). Blots were then incubated with rabbit (sc-2313, Santa Cruz, TX, USA) or mouse (A9044, Sigma-Aldrich, MO, USA) horseradish peroxidase-conjugated secondary antibody diluted to 1:4000 depending on primary antibody species. Blots were developed using the ECL™ Prime Western Blotting System (GE Healthcare, IL, USA) on the Chemidoc Touch Imaging System (Bio-Rad, CA, USA). Densitometry was performed using Bio-Rad Image Lab 5.2.1 (Bio-Rad, CA, USA).
Immunofluorescence staining
Cells cultured in chamber slides were fixed with 2% paraformaldehyde for 15 min at room temperature and permeabilised with 0.1% Triton X-100. Samples were blocked in PBS with 10% goat serum and 1% BSA for 1 h. Samples were incubated with primary MYCN antibody SC-53993 (1:500, Santa Cruz, TX, USA) overnight at 4 °C followed by secondary antibody Alexa Fluor 555 goat anti-mouse (1:400, Invitrogen, CA, USA) for 30 min at room temperature. Cells were counterstained with DAPI. Fluorescent images were captured using a Zeiss Axioplan 2 microscope (Oberkochen, Germany) using a 16× objective and a standard exposure time optimised for control treated cells. The sum of the intensity for MYCN staining was measured using Image J software and made relative to the number of cells in that field of view, indicated by DAPI.
Plasmid production and transfection
Full-length MYCNOS-01 transcript (RefSeq NR_110230) was cloned into the pcDNA5/TO vector (Invitrogen, CA, USA) and the construct verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). For plasmid transfection, Lipofectamine 2000 (Invitrogen, CA, USA) was used following the manufacturer’s instructions. Samples were analyzed by qRT-PCR and Western blot at time-points indicated in the relevant figures.
Proteasome inhibition
For protein stability experiments, cells were transfected for a total of 48 h and treated for the final 4 h with either 10 μM MG132 (Sigma-Aldrich, MO, USA) in DMSO or DMSO control. Protein was then extracted from cells for analysis by Western blot.
Cell viability assay
Cells were transfected as six repeats in a 96-well plate to assess the effects of gene knockdown. Cell viability was assessed by the MTS method using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, WI, USA). Fresh media plus 20 μl assay reagent were added at the indicated time-point. After 2.5 h incubation at 37 °C and 5% CO2 the absorbance of each well was measured at 492 nm on a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany).
Apoptosis assay
Cells were transfected in quadruplicate in a 96-well plate and apoptosis measured by evaluating the activation of caspase 3/7 at the indicated time-point by replacement of 50 μl of media with 50 μl of Caspase-Glo® 3/7 Assay (Promega, WI, USA). After 1 h incubation protected from light at room temperature, the samples were transferred to a white-walled 96-well plate and luminescence of each well was read on a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany). The caspase signal intensity was normalised by the absorbance measurement from the corresponding MTS assay.
Flow cytometry
Cells were fixed in 70% ethanol at -20 °C overnight, washed with PBS and resuspended in 1 ml PBS containing 100 μg/ml RNase A and 40 μg/ml Propidium Iodide. Samples were incubated for 30 min at 37 °C then analyzed on a BD™ LSRII Flow Cytometer (BD Biosciences, CA, USA).
Statistical analysis
Graphs represent means ± standard deviation from multiple independent experiments as stated in figure legends. Statistical significance was measured by unpaired two-tailed Student t-test or by one-way analysis of variance (ANOVA) with post hoc Dunnett’s test for multiple comparisons. For linear correlation studies of gene expression Pearson’s coefficient (R) was calculated between each pair of variables to indicate the strength of the linear association. p < 0.05 was considered significant and indicated by a single asterisk, p < 0.01 is indicated by a double asterisk, and p < 0.001 is indicated by a triple asterisk.
Discussion
In this study, we have shown that
MYCNOS-01 and
MYCNOS-02 can play important roles in RMS as well as NB cell growth, at least in part via their regulation of MYCN. Roles for
MYCNOS-01 in RMS and NB and
MYCNOS-02 in RMS have not been previously explored whilst our data for the effect of
MYCNOS-02 on growth of NB cells is consistent with previous findings [
17‐
22]. Regulation of MYCN by
MYCNOS-01 and
MYCNOS-02 was readily apparent in
MYCN-amplified RMS and NB, which express these transcripts at high levels, presumably as a result of their co-amplification at the genomic level. In contrast, effects of
MYCNOS transcripts on MYCN protein levels in RMS and NB without high level
MYCN amplification were either less marked or not seen. The positive regulation of MYCN by
MYCNOS-01 and
MYCNOS-02 likely contribute to the cell growth of RMS and NB. This is consistent with the phenotypic dose dependent effects and dependencies of RMS and NB cells on MYCN levels seen in this and previous studies [
9,
29,
30].
In addition to the positive regulation of MYCN by
MYCNOS-01, MYCN also negatively regulates
MYCNOS-01 transcription, summarized in Fig.
7. As MYCN protein has been found to be recruited to its own intron 1 [
10], in line with the transcription start site for
MYCNOS-01 on the opposite strand, this binding activity could be involved in MYCN-mediated regulation of
MYCNOS-01. Previous studies have also identified indirect MYCN negative feedback mechanisms involving
trans-acting factors [
12]. This is likely a mechanism that fine-tunes MYCN expression levels.
An increasing number of lncRNAs have now been characterized and many have been linked to cancer progression [
31]. Here we have shown that
MYCNOS-01 can act as a
cis-antisense lncRNA on its sense partner MYCN. Although
MYCN transcript expression was not regulated by
MYCNOS-01, we have identified a post-transcriptional role for this lncRNA in regulating MYCN protein levels. This is consistent with the lack of correlation we found between the two transcripts in non-amplified lines.
There are several examples of lncRNAs that regulate protein partners post-transcriptionally without affecting transcript expression [
32‐
35]. For example, the lncRNA
PVT1 has been shown to be required for increasing MYC protein stability and high expression levels in 8q24-amplified cancers [
32]. In our study, we found no evidence that
MYCNOS-01 altered the stability of MYCN protein via Thr58/Ser62 phosphorylation. Another example of post-transcriptional regulation is the lncRNA
treRNA, which has been shown to play a role in tumor invasion and metastasis in breast cancer, and which regulates the translation of E-cadherin mRNA in these cells via redistribution of
CDH1 to low molecular weight polysomes to suppress translation [
34]. Potentially
MYCNOS-01 could have a similar mechanism to regulate translation efficiency of MYCN and thus its protein expression. In addition, the lncRNA
BACE1-AS regulates translation of
BACE1 by masking the binding site for miR-485-5p
, thus preventing miRNA-induced translational repression and mRNA decay [
35]. Another possibility therefore is that
MYCNOS-01 interacts with an miRNA that targets MYCN for degradation, therefore increasing MYCN protein expression by sequestering away a negative regulating factor. The above examples indicate possible post-transcriptional mechanisms that could be explored for
MYCNOS-01-mediated regulation of MYCN. Further investigations are required to determine molecular interactions with
MYCNOS-01 and how these regulate MYCN protein levels.
Both
MYCNOS-01 and
MYCNOS-02 shown in this study in RMS and NB, and
MYCNOS-02 shown previously in NB, regulate MYCN protein levels [
17,
19,
20]. However, data on whether
MYCNOS-02 is able to regulate MYCN at the transcriptional level is conflicting. One report for
MYCNOS-02 indicates silencing of
MYCNOS-02 does not affect
MYCN expression at the transcriptional level [
17]. However, other studies suggest that
MYCNOS-02 can affect
MYCN transcript expression in NB [
18,
19,
21].
MYCNOS-02 has been found to interact with CTCF to affect chromatin remodeling at the
MYCN promoter therefore
MYCN transcript expression [
19]. However, in silico prediction techniques suggest CTCF interacts with a region of
MYCNOS-02 that does not overlap with the sequence of
MYCNOS-01, supporting the possibility that the two transcripts could have different binding partners that are involved in MYCN regulation. It is possible for two overlapping lncRNAs from the same locus to have different characteristics and function. For example, the
CCAT1 locus encodes
CCAT1-L that is located in the nucleus and positively regulates
MYC transcription and
CCAT1-S that is mainly located in the cytoplasm with no effect on
MYC transcript levels [
36‐
38].
Previous studies of
MYCNOS-02 have identified its role in NB tumor growth and metastasis. In vitro
, MYCNOS-02 can suppress differentiation and promote metastasis, invasion and cell proliferation partially due to its indirect regulation of MYCN [
19]. Our study found
MYCNOS-01 also plays a role in
MYCN-amplified RMS and NB cell viability, although no effect was seen on cell cycle progression. Often a decrease in proliferation occurs with concomitant cell cycle arrest, however it is possible for these two effects to be separated. For example, one study found decreasing the tumor suppressors RPL5 or RPL11 resulted in a reduction in ribosome content and translation capacity, causing cells to progress at a lower rate through all stages of the cell cycle thus resulting in decreased proliferation without cell cycle arrest [
39].
MYCNOS-01 was also found to play a role in cell growth via regulation of MYCN; silencing of
MYCNOS-01 resulted in a reduction in MYCN protein levels. However,
MYCNOS-01 knockdown did produce a slightly different phenotype to
MYCN knockdown due to differences in MYCN protein levels achieved. We have previously identified that using different molecular tools to diminish MYCN can affect the strength of phenotype detected depending on the magnitude and endurance of MYCN reduction [
9].
MYCNOS-01 knockdown did not decrease MYCN protein sufficiently to produce a G1 arrest, in contrast to direct
MYCN knockdown. However,
MYCNOS-01 reduction affecting other protein targets and signaling pathways that contribute to the phenotype observed cannot be excluded. Further defining how
MYCNOS-01 regulates MYCN, and possibly other proteins, may lead to new approaches to perturb the clinically aggressive phenotype of RMS and NB tumors.