The long noncoding RNA LINC00200 promotes the malignant progression of MYCN-amplified neuroblastoma via binding to insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) to enhance the stability of Zic family member 2 (ZIC2) mRNA.

Neuroblastoma (NB) is one of the most common extracranial malignant tumors in children and remarkable heterogeneous tumor of the sympathetic nervous system. Long noncoding RNAs (lncRNAs) have been reported to be vital biological roles in the initiation and malignant progression of tumors. The aim of this study was to explore the biological role and the possible molecular mechanism of LINC00200 in NB. The expression level of LINC00200 in NB tissues and cell lines were detected by real-time quantitative PCR (qRT-PCR). The biological effects of LINC00200 on NB cell proliferation, migration and invasion were examined by EdU and transwell assays. The molecular mechanism of LINC00200 in NB were explored and verified by bioinformatics analysis, RNA binding protein immunoprecipitation (RIP) assay and RNA pull down assay. The results showed that the expression of LINC00200 was significantly higher in NB tissues than in normal tissues. Besides, the expression of LINC00200 was higher in MYCN Amplified NB tissues than in MYCN non-Amplified NB tissues. Moreover, overexpression of LINC00200 could remarkedly promote proliferation, migration and invasion of NB cell. Mechanistically, LINC00200 might bind to RNA binding protein (RBP) IGF2BP3 and promote the expression of ZIC2. Overall, we showed that LINC00200 was upregulated in NB tissues and the LINC00200/IGF2BP3/ZIC2 regulatory axis might be the possible therapeutic target for NB.

Chen J ，Sun M ，Huang L ，Fang Y ... -  《-》

LncRNA AC142119.1 facilitates the progression of neuroblastoma by epigenetically initiating the transcription of MYCN.

Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear. Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter. AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression. We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.

Yang R ，Liu N ，Li T ，Liu F ，Zhang J ，Zhao H ，Zou L ，He X ... -  《Journal of Translational Medicine》

CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

Zhao X ，Li D ，Pu J ，Mei H ，Yang D ，Xiang X ，Qu H ，Huang K ，Zheng L ，Tong Q ... -  《-》

KAP1 stabilizes MYCN mRNA and promotes neuroblastoma tumorigenicity by protecting the RNA m(6)A reader YTHDC1 protein degradation.

Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

Yang Y ，Zhang Y ，Chen G ，Sun B ，Luo F ，Gao Y ，Feng H ，Li Y ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

miR-15a-5p, miR-15b-5p, and miR-16-5p inhibit tumor progression by directly targeting MYCN in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid malignancy in children. Despite current aggressive treatment regimens, the prognosis for high-risk NB patients remains poor, with the survival of less than 40%. Amplification/stabilization of MYCN oncogene, in NB is associated with a high risk of recurrence. Thus, there is an urgent need for novel therapeutics. The deregulated expression of microRNA (miR) is reported in NB; nonetheless, its effect on MYCN regulation is poorly understood. First, we identified that miR-15a-5p, miR-15b-5p, and miR-16-5p (hereafter miR-15a, miR-15b or miR-16) were down-regulated in patient-derived xenografts (PDX) with high MYCN expression. MiR targeting sequences on MYCN mRNA were predicted using online databases such as TargetScan and miR database. The R2 database, containing 105 NB patients, showed an inverse correlation between MYCN mRNA and deleted in lymphocytic leukemia (DLEU) 2, a host gene of miR-15. Moreover, overexpression of miR-15a, miR-15b or miR-16 significantly reduced the levels of MYCN mRNA and N-Myc protein. Conversely, inhibiting miR dramatically enhanced MYCN mRNA and N-Myc protein levels, as well as increasing mRNA half-life in NB cells. By performing immunoprecipitation assays of argonaute-2 (Ago2), a core component of the RNA-induced silencing complex, we showed that miR-15a, miR-15b and miR-16 interact with MYCN mRNA. Luciferase reporter assays showed that miR-15a, miR-15b and miR-16 bind with 3'UTR of MYCN mRNA, resulting in MYCN suppression. Moreover, induced expression of miR-15a, miR-15b and miR-16 significantly reduced the proliferation, migration, and invasion of NB cells. Finally, transplanting miR-15a-, miR-15b- and miR-16-expressing NB cells into NSG mice repressed tumor formation and MYCN expression. These data suggest that miR-15a, miR-15b and miR-16 exert a tumor-suppressive function in NB by targeting MYCN. Therefore, these miRs could be considered as potential targets for NB treatment.

Chava S ，Reynolds CP ，Pathania AS ，Gorantla S ，Poluektova LY ，Coulter DW ，Gupta SC ，Pandey MK ，Challagundla KB ... -  《-》