The Identification And The Role Of NcRAN, A Novel Non-coding RNA Mapped To Chromosome 17q In Neuroblastoma

PrefaceNeuroblastoma shows complex patterns of genetic aberrations including MYCN amplification,deletion of chromosome 1p or 11q,and gain of chromosome 17q.The 17q gain is frequently observed in high-risk neuroblastomas,however,the candidate genes still remain elusive.To find them out,we integrated the data of comparative genomic hybridization of 236 tumors by BAC array and expression profiling of 136 tumors by using the in-house cDNA microarray carrying 5,340 genes derived from primary neuroblastomas.Here we have identified a novel candidate gene mapped to chromosome 17q25.1 with two splicing variants,Nblal0727 and Nblal2061.The transcript size appeared to be 2.3kb by Northern blot,however,the cDNA sequences had no obvious open reading frame. The protein product was undetectable by both in vivo and in vitro translation assays, suggesting that the transcript might not encode any protein product.Therefore,we named it as ncRAN(non-coding RNA expressed in aggressive neuroblastoma).In analysis of 70 patients with sporadic neuroblastoma,the high levels of ncRAN mRNA expression were significantly associated with poor outcome of the patients (p＜0.001).The multivariate analysis showed that expression of ncRAN mRNA was an independent prognostic factor from age,stage,origin and MYCN expression.Ectopic expression of ncRAN induced transformation of NIH3T3 cells in soft agar,while knockdown of endogenous ncRAN with RNA interference significantly inhibited cell growth in SH-SY5Y cells.Taken together,our results suggest that ncRAN may be a novel non-coding RNA mapped to the region of 17q gain and act like an oncogene in aggressive neuroblastomas.Materials and methods1,Patients.Tumor specimens were collected from the patients with neuroblastoma who had undergone biopsy or surgery at various institutions in Japan.Two hundreds and thirty-six and 136 tumor samples were used for array-CGH and expression profiling,respectively.Among them,sporadic cases were 112 and 70,respectively.The clinical stage of tumor was classified according to the INSS criteria.Expression data for the latter 70 sporadic neuroblastomas,which were composed of 15 stage 1,8 stage 2,17 stage 3,25 stage 4,and 5 stage 4s tumors,were used for the Kaplan-Meier analysis.The status of MYCN amplification in each tumor had been determined as described previously.Patients were treated according to previously described protocols. The procedure of this study was approved by the Institutional Review Board of the Chiba Cancer Center(CCC19-9).2,Microarray-based comparative genomic hybridization(array-CGH) and gene expression profiling.Array-based CGH experiments for 236 neuroblastomas by using a chip carrying 2,464 BAC clones which covers the whole human genome at roughly 1.2-Mb resolution were performed as described previously.For the gene expression profiling of 136 neuroblastomas,we employed an in-house cDNA microarray,carrying 5,340 cDNAs obtained from the oligo-capping cDNA libraries generated from anonymous neuroblastoma tissues.The array-CGH and gene expression profile data are available at NCBI Gene Expression Omnibus(http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE 5784 and GSE 5779,respectively. 3,Cells,culture and transfection.NIH3T3,COS7 and human neuroblastoma cell lines were cultured in Dulbecco modified Eagle medium(DMEM) or RPMI 1640 medium containing 10%(vol/vol) heat-inactivated fetal bovine serum(FBS) and antibiotics.Cultures were maintained in a humidified atmosphere containing 5%CO₂ at 37℃.COS7 and NIH3T3 cell lines were transiently transfected using Lipofectamine 2000 reagent(Invitrogen;Carlsbad,USA) according to the manufacturer's protocol.4,Construction of expression plasmid.The full-length cDNAs of Nbla10727 and Nbla12061 were cloned from the established full length-enriched cDNA libraries which we made from the primary neuroblastomas as described.The full-length cDNAs were then inserted into pcDNA3 or pcDNA3-FLAG plasmids.5,In vitro transcription and translation assay.In vitro translation was carried out in the presence of[³⁵S]-methionine using TNT T7 Quick Coupled Transcription/Translation System(Promega,Madison,WI,USA) according to the manufacturer's instructions.The products were resolved by SDS-PAGE and detected by autoradiography.6,In vivo[³⁵S]labeling experiment.COS7 cells were transfected with the FLAG-tagged ncRAN expression vectors or the HA-tagged MEL1 expression plasmid. After 24 hours,cells were rinsed with 1×PBS 3 times and recultured in fresh growth medium without methionine and antibiotics.Two hours later,[³⁵S]-methionine(GE Healthcare,Tokyo,Japan) was added to the medium to a final concentration of 0.1mCi/ml,and cells were further incubated.Cells were harvested and whole cell lysates were subjected to immunoprecipitation using a monoclonal anti-Flag antibody or a polyclonal anti-HA antibody.Immunoprecipitates were resolved by SDS-PAGE and detected by autoradiograph.7,RNA isolation and semi-quantitative reverse transcription-PCR(RT-PCR).Total RNA was isolated from frozen tumor tissues by an AGPC method.Five micrograms of total RNA were employed to synthesize the first-strand cDNA by means of random primers and Superscriptâ…¡reverse transcriptase(Invitrogen,California,USA) following the manufacture's protocol.We prepared appropriate dilutions of each single stranded cDNA for subsequent PCR by monitoring an amount of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as a quantitative control.The PCR amplification was carried out for 28 cycles(preheat at 95℃for 2 min,denature at 95℃for 15s,annealing at 55℃15s,and extension at 72℃20s) for ncRAN(Nbla10727 and Nbla12061).The primers used were as follows:ncRAN(Nbla10727) 5'-CAGTCAGCCTCAGTTTCCAA-3'(forward);5'-AGGCAGGGCT GTGCTGAT-3' (reverse),ncRAN(Nbla12061) 5'-ATGTTAGCTCCCAGCGAT GC-3'(forward); 5'-CTAACTGCCAAAAGGTTTTCC-3'(reverse).8,Northern Blot Analysis.Twentyμg of total RNA was subjected to electrophoresis and Northern blotting.The cDNA insert(Nbla10727) was labeled with [32^P]-dCTP(GE Healthcare,Tokyo,Japan) by the BcaBEST^TM labeling Kit(Takara, Tokyo,Japan) and used for the hybridization probe.9,Soft agar assay.NIH3T3 cells were transfected with FLAG-Nbla10727, FLAG-Nbla12061 or empty vector,and resuspended in 0.33%agar(wt/vol) in DMEM with 10%FBS at a density of 500 cells/plate.Cell suspensions were poured on the top of the base layer(0.5%agar(wt/vol) in fresh medium,and grew in a 5%CO₂ incubator for 14 days.Colonies larger than l00μm were counted under an Olympus microscope.10,RNA interference.Oligonucleotides for knocking down the ncRAN with Sac-I and Xho-I extension were inserted into pMuni vector.The oligonucleotides used are as follows:5'-CCCCATCCTCTAGTAGCCACGGTTTCAAGAGAACCGTGGCTACTA GAGGATTTTTTGGAAAC-3' and 5'-TCGAGTTTCCAAAAAATCCTCTAGTA GCCACGGTTCTCTTGAAACCGTGGCTACTAGAGGATGGGGAGCT-3'.The plasmids containing the oligonucleotide sequence were transfected into SH-SY5Y cells by using Lipofectamine 2000 reagent(Invitrogen;Carlsbad,CA) according to the manufacturer's protocol.11,Statistical analysis.The Student's t-tests were used to explore possible associations between ncRAN expression and other factors,such as age.Kaplan-Meier curves were calculated and survival distributions were compared using the log-rank test. Univariate and multivariate analyses were made according to the Cox hazard models. q-value was also calculated because ncRAN expression was measured with 5,340 genes in the microarray.Statistical significance was declared if the p-value was＜0.05.Results1,Identification of a novel gene mapped to chromosome 17q25.1 upregulated in advanced neuroblastomas with gain of chromosome 17q.To explore the candidate genes for therapeutic target against aggressive neuroblastomas,the genomic and molecular characteristics specific to high-risk tumors were surveyed.We previously conducted array-CGH analysis with a microarray carrying 2,464 BAC clones to examine genomic aberrations in 236 primary neuroblastomas and found that the gain of chromosome 17q was most strongly correlated with the patient's prognosis. Consequently,we found two cDNA clones Nblal0727 and Nblal2061 on our in-house microarray carrying 5,340 cDNAs obtained from oligo-capping cDNA libraries generated from different subsets of primary neuroblastomas,both of which were splicing variants of the same gene mapped to chromosome 17q25.12,High expression of ncRAN-Nbla10727/12061 is associated with poor prognosis of neuroblastoma.The analysis by semi-quantitative RT-PCR in a panel of cDNAs obtained from 8 favorable(stage1,less than 1-year-old,single copy of MYCAN and high expression of TrkA) and 8 unfavorable(stage 3 or 4,more than 1-year-old,amplified MYCN and low expression of TrkA) primary neuroblastomas confirmed that this novel gene was expressed significantly at high levels in the latter than the former,like Survivin which we have previously reported as one of the candidate genes mapped at the region of 17q gain.As expected,among neuroblastoma cell lines,high or moderate levels of expression of ncRAN-Nbla10727/12061 was observed in cell lines withMYCN amplification most of which had 17q gain,whereas it was relatively low in those with a single copy of MYCNand without the 17q gain.Our microarray data of 70 sporadic neuroblastomas showed that the high levels of ncRAN-Nbla10727/12061 expression were significantly associated with poor prognosis (log-rank test,p=0.000221 and p=0.005728,respectively).The multivariate analysis using Cox proportional hazard model demonstrated that expression of ncRAN-Nbla10727/12061 was an independent prognostic factor from age at diagnosis, disease stage,tumor origin and MYCN expression.Thus,the expression level of ncRAN-Nbla10727/12061 is a novel prognostic factor of neuroblastoma that is closely associated with gain of chromosome 17q.3,ncRAN-Nbla10727/12061 is involved in inducing enhancement of cell growth in neuroblastoma cells and transformation of NIH3T3 cells.To investigate function of ncRAN-Nbla10727/12061,we transfected SH-SY5Y neuroblastoma cells with the siRNA,since SH-SY5Y cells have 17q gain in their genome as well as higher mRNA expression of ncRAN-Nbla10727/12061.Suppression of endogenous levels of Nbla10727/12061 transcripts significantly inhibited cell growth in SH-SY5Y neuroblastoma cells as compared with the control cells.On the other hand,the soft agar colony formation assay showed that the enforced expression of ncRAN-Nbla10727/12061 significantly enhanced the anchorage-independent growth of NIH3T3 mouse fibroblast cells.These results suggested that ncRAN-Nbla10727/12061 was a novel candidate gene of the region of 17q gain with an oncogenic function.4,ncRAN-Nbla10727/12061 is a large non-coding RNA.Several lines of evidences from the gene structure analysis as well as the comparative genomic analysis described below further suggested that ncRAN-Nbla10727/12061 is a non protein-coding but functional RN² e therefore tentatively named this gene as ncRAN(non-coding RNA expressed in aggressive neuroblastoma).First,the full-length cDNA sequences of ncRAN,which are suggested to be relevant to both Nblal0727 and Nblal2061 cDNAs by Northern blot analysis, contained no long enough open reading frames(＞200-bp).Bioinformatic analysis indicated that there were no ESTs longer than those two cDNAs at the genomic locus, and that the CpG island was located at the 5' region of the cDNA sequences.Second,no protein product was translated both in vivo and in vitro from the ncRAN transcripts.Though only the possible open reading frames(＞150-bp) within the ncRAN cDNA were from n.t.190 to 354(55 amino acids) and from 293 to 469(59 amino acids) in Nbla10727,none of the putative translation start sites contains the Kozak consensus sequence.In addition,these predicted protein products of 55 and 59 amino acids did not at all exhibit significant similarity to any other known protein or protein domain. Furthermore,in vivo transcription and translation of the full-length ncRAN did not lead to the synthesis of any peptide or protein,though endogenously and ectopically expressed ncRAN could be easily detectable at mRNA level.Coincident with the above observation,none of the ncRAN protein product could be detected using [³⁵S]-methionine-labeling system in vitroThird,we performed sequence comparison of the ncRAN gene with genome sequences of other species and found its high similarities(＞90%identity in nucleotides) with primates including orangutan,chimpanzee and rhesus,but not those with mice and rat.We also searched for the possible long open reading frames of ncRAN homologs in these highly similar species,resulting in failure.The highly conserved sequence similarity only with primates may suggest that ncRAN might be an evolutionally developed non-coding RNA.Finally,previous studies have shown that some large non-coding RNAs are relevant to host RNAs that harbor small RNAs such as microRNA(miRNA).Therefore, we made a search for sequences of known miRNAs in conserved regions within the ncRAN locus,but none were identified so far.These results inferred that the ncRAN transcript might not be processed to one or more small RNAs.In addition,database search did not identify genes with anti-direction to ncRAN,excluding the possibility that ncRAN is an antisense gene for certain known genes.Taken together,these results strongly suggested that the ncRAN transcript functions as a novel large non-coding RNA.Conclusions1,ncRAN is a novel non-coding RNA mapped to chromosome 17q25.1 in neuroblastoma.2,The high expression of ncRAN is associated with poor prognosis of neuroblastoma.3,ncRAN with characters of oncogen is a large non-coding RNA.