Dataset Information

Long Non-coding RNA SNHG1 in Neuroblastoma

ABSTRACT: Neuroblastoma (NB) is an embryonal tumor with various clinical presentations and behaviors. Several genomic alterations has been well-studied in NB, among which genomic amplification of MYCN oncogene, is a strong prognostic biomarker with worsens outcome. Long noncoding RNAs (lncRNAs), constitute major proportion of the cellular transcripts with no coding capacity. One of their function is to guide transcription factors to the target genes and facilitate gene expression. However, relative contribution of lncRNA and MYCN to the advanced NB has remained unclear. Herein, by applying a network-based integrative analysis on MYCN amplified and MYCN nonamplified lncRNA expression profile from both RNA-seq and microarray platform, we identified lncRNA, SNHG1 to be differentially expressed and strongly correlated with MYCN in MYCN-amplified NB. The expression of SNHG1 was validated by RT-qPCR in NB cell lines. Survival analysis revealed that higher expression of SNHG1 significantly associates with poor patient survival. Moreover, knockdown of MYCN in MYCN-amplified NB cell lines inhibited SNHG1 expression. Furthermore, to unravel the role of SNHG1 in NB, we extracted SNHG1-interacting proteins by RNA-protein pull down assay coupled with doi:10.6342/NTU201701980 ! ! VI liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 27 SNHG1-interacting proteins in common from three NB cell lines. However, only three SNHG1-interacting proteins, MATR3, YBX1 and HHRNPL have binding site detected by DeepBind motif analysis. Western blot confirms interaction of MATR3 with SNHG1. Additionally, we further validated the direct interaction between MATR3 and SNHG1 by RNA-immunoprecipation (IP). MATR3 is known to be involved in RNA transport and stabilization. Therefore, we proposed that MATR3 after interacting with SNHG1 might help in SNHG1 transcription and stabilization. In conclusion, our study unveils that SNHG1 could be a prognostic marker for high-risk NB and possibly stabilized by MATR3. Our results might provide future directions for the development of therapeutic strategies against high-risk NB.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Neuroblastoma

SUBMITTER: Tz-Wen Yang

LAB HEAD: Hsueh-Fen Juan

PROVIDER: PXD007121 | Pride | 2022-03-01

REPOSITORIES: Pride

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			Action	DRS
	170302MS11_0222_BE_Ctrl_1.raw	Raw
	170302MS12_0222_BE_Ctrl_2.raw	Raw
	170302MS14_0222_AS_1.raw	Raw
	170302MS15_0222_AS_2.raw	Raw
	170302MS17_0222_AS_Ctrl_1.raw	Raw

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Publications

RNA-Binding Proteomics Reveals MATR3 Interacting with lncRNA SNHG1 To Enhance Neuroblastoma Progression.

Yang Tz-Wen TW Sahu Divya D Chang Yi-Wen YW Hsu Chia-Lang CL Hsieh Chiao-Hui CH Huang Hsuan-Cheng HC Juan Hsueh-Fen HF

Journal of proteome research 20181214 1

The interaction of long noncoding RNAs (lncRNAs) with one or more RNA-binding proteins (RBPs) is important to a plethora of cellular and physiological processes. The lncRNA SNHG1 was reported to be aberrantly expressed and associated with poor patient prognosis in several cancers including neuroblastoma. However, the interacting RBPs and biological functions associated with SNHG1 in neuroblastoma remain unknown. In this study, we identified 283, 31, and 164 SNHG1-interacting proteins in SK-N-BE( ...[more]

PMID: 30516047

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Project description:Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with diverse clinical behaviors. Even with multimodal therapies, high-risk neuroblastoma has an unfavorable outcome irrespective of MYCN amplification, a well-established oncogenic driver in neuroblastoma pathogenesis, and its genetic heterogeneity has largely impeded efforts to correlate molecular targets with biological consequences for more effective treatment strategies. Here, using a gene expression-based approach, we identified the FDA-approved anthelmintic niclosamide as a potential anti-neuroblastoma drug. By combining the gene expression signature associated with high-risk neuroblastoma and the recurrent drug−transcript relationships inferred from up to one million perturbational gene expression profiles, our algorithm predicted effective therapeutic candidates by evaluating the extent to which a given compound or their combinations could ‘reverse’ the high-risk signature. Furthermore, we performed quantitative polymerase chain reaction (qPCR) to validate top five candidate reverse genes which are involved in DNA replication, including cyclin A2 (CCNA2), minichromosome maintenance 10 replication initiation factor (MCM10), ERCC excision repair 6 like, spindle assembly checkpoint helicase (ERCC6L), kinesin family member 20A (KIF20A), and RuvB like AAA ATPase 1 (RUVBL1). Indeed, those five genes were downregulated in niclosamide-treated cells, indicating niclosamide suppressed DNA replication and then inhibited cell proliferation. Using cell proliferation and clonogenic assays as well as flow cytometry, we determined the cytotoxic effects of niclosamide in MYCN-amplified SK-N-DZ and non-amplified SK-N-AS cells. The results showed that niclosamide could effectively reduce not only cell proliferation and colony formation but also trigger cell cycle arrest and apoptosis. Moreover, we conducted human tumor xenografts in a nude mice model to evaluate the in vivo efficacy of niclosamide and found that it significantly suppressed tumor growth and prolonged survival rate, but doesn’t cause organ damage and change body weight. To explore the molecular mechanism of niclosamide, stable-isotope dimethyl labeling strategy for quantitative proteomics was performed on both cell-based or xenograft-based MYCN-amplified SK-N-DZ and MYCN-nonamplified SK-N-AS models. We confirmed niclosamide not only mediated the function of mitochondrial electron transport chain but also the other functions in high risk neuroblastoma cell lines and xenografts. The results suggest that our developed expression-based strategy is useful for drug discovery and provides the possibility of repurposing the anthelminthic drug niclosamide for treating high-risk neuroblastoma therapy.

Project description:Simple Summary: Neuroblastoma (NB) accounts for 15% of all cancer related deaths of children. While amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB, expression of the neurotrophin receptor, NTRK1/TrkA, has been shown to associate with an excellent outcome. MYCN down-regulates NTRK1 expression, but it is unknown if the molecular effects of NTRK1 signaling also affect MYCN-induced networks. The aim of this study was to decipher NTRK1 signaling using an unbiased proteome and phosphoproteome approach. To this end, we realized inducible ectopic NTRK1-expression in a NB cell line with MYCN amplification and analyzed the proteomic changes upon NTRK1-activation in a time-dependent manner. In line with phenotypes observed, NTRK1-activation induced markers of neuronal differentiation and cell cycle arrest. Most prominently, NTRK1 upregulated expression and phosphorylation of the nuclear lamina component Lamin A/C. Moreover, NTRK1 signaling also induced aggregation of LMNA within nucleic foci, which accompanies differentiation in other cell types. Abstract: Background: Neuroblastomas (NB) are the most common extracranial solid tumors of childhood. Amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. Methods: Inducible NTRK1-expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1-activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1-targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. Phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.