Neuroblastoma Tissues Research Articles

Downregulation of lncRNA XIST Represses Tumor Growth and Boosts Radiosensitivity of Neuroblastoma via Modulation of the miR-375/L1CAM Axis

Neuroblastoma (NB) is a heterogeneous tumor that is common in infants and young children. Long non-coding RNA X-inactive specific transcript (XIST) is implicated in NB advancement. Nevertheless, the role and regulatory mechanism by which XIST in NB are not fully elucidated. Expression levels of XIST, microRNA-375-5p (miR-375), and L1 cell adhesion molecular (L1CAM) were examined through quantitative real-time polymerase chain reaction (qRT-PCR). The cell cycle progression, proliferation, and colony formation of NB cells were determined with flow cytometry, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), or cell colony formation assays. Cell apoptotic rate was detected with flow cytometry assay. The relationship between XIST or L1CAM and miR-375 was verified via dual-luciferase reporter assay. The level of L1CAM protein was examined through western blotting. The role of XIST in vivo was confirmed through xenograft assay. XIST and L1CAM were upregulated while miR-375 was downregulated in NB tissues and cells. XIST depletion repressed tumor growth in vivo and elevated radiosensitivity, arrested cell cycle progression, and impeded proliferation of NB cells in vitro. Mechanistically, XIST modulated L1CAM expression through competitively binding to miR-375. Furthermore, miR-375 inhibitor recovered XIST inhibition-mediated effects on the radiosensitivity and malignant behaviors of NB cells. Also, L1CAM overexpression reversed the effects of miR-375 enhancement on the cell cycle progression, proliferation, and radiosensitivity of NB cells. XIST downregulation repressed tumor growth and boosted radiosensitivity of NB via modulating the miR-375/L1CAM axis, indicating that XIST was a promising target for NB treatment.

Abstract 5721: The super-enhancer driven long noncoding RNA lncNB promotes neuroblastoma tumorigenesis

Abstract Background: While MYCN-amplified neuroblastoma has been the focus of neuroblastoma research in the past three decades, most human neuroblastomas do not harbour MYCN oncogene amplification, and their tumorigenic factors are unknown. The expression of critical oncogenes is driven by transcriptional super-enhancers which are controlled by the BET bromodomain protein BRD4 and the transcription factor TFIIH-associated kinase CDK7. Long noncoding RNAs (lncRNAs) play important roles in cancer. Aims: To determine the mechanism through which the novel lncRNA lncNB is up-regulated in MYCN-non-amplified neuroblastoma; to determine the role of lncNB in neuroblastoma cell proliferation, survival and tumour progression; and to identify the mechanism through which lncNB exerts oncogenic effects. Methods and results: RNA sequencing analysis identified lncNB as one of the transcripts most dramatically over-expressed in MYCN non-amplified neuroblastoma cell lines and tumour tissues, and Kaplan Meier analysis showed that high levels of lncNB expression in neuroblastoma tissues correlated with poor prognosis in 476 patients. Examination of published chromatin immunoprecipitation sequencing data revealed super-enhancers at the lncNB gene locus only in MYCN non-amplified neuroblastoma cells. RT-PCR experiments showed that transfection with two independent BRD4 siRNAs or treatment with the BRD4 inhibitor AZD5153 or the CDK7 inhibitor THZ1 considerably reduced lncNB expression and cell proliferation in MYCN non-amplified neuroblastoma cells. Alamar blue assays, cell cycle analysis, BRDU assay and clonogenic assays revealed that lncNB knockdown with siRNAs or doxycycline-inducible shRNAs in MYCN non-amplified neuroblastoma cells led to growth inhibition, apoptosis and dramatic reduction in clonogenic capacity. RNA-binding protein pull-down and RNA immunoprecipitation assays identified MSI as a binding partner for the lncNB lncRNA; and RNA sequencing has identified BMX as the downstream target of lncNB. In addition, Balb/C nude mice were xenografted with doxycycline-inducible lncNB shRNA MYCN non-amplified neuroblastoma cells. Knocking down lncNB with doxycycline in food considerably suppressed tumour progression and improved mouse survival. Conclusions: The data suggest that the lncRNA lncNB is upregulated by super-enhancer activity in MYCN non-amplified neuroblastoma cells, that lncNB up-regulation induces neuroblastoma cell proliferation, survival, clonogenic capacity and tumorigenesis, and that targeting lncNB expression can be an effective therapeutic strategy. Citation Format: Sujanna Mondal, Matthew Wong, Andrew Tee, Nicholas Ho, David S. Ziegler, Glenn M. Marshall, Pei Y. Liu, Marcel Dinger, Tao Liu. The super-enhancer driven long noncoding RNA lncNB promotes neuroblastoma tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5721.

RNA N6-methyladenosine modification is required for miR-98/MYCN axis-mediated inhibition of neuroblastoma progression

Neuroblastoma (NB) is one of the most common malignant tumors of the sympathetic nervous system in childhood. NB severely threatens patient’s health and life. However, more effective diagnosis and treatment methods are badly needed in clinics all over the world. MYCN is well recognized as a genetic biomarker of high risk and poor outcome in NB. miRNAs are small RNAs and miR-98 involved in the pathogenesis of various cancers. The role and mechanism of miR-98 in NB remains to be investigated. Here we found that miR-98 was decreased in human MYCN-high-expression NB tissues, and its down-regulation was associated with poor prognosis of NB. Over-expression of miR-98 inhibited cell proliferation, migration and invasion of NB cells. The analysis by employing the software of miRanda predicted the possible binding sites of miR-98 in the 3′-UTR of MYCN, and experimental data illustrated that miR-98 directly bound to MYCN 3′-UTR and decreased MYCN expression. Over-expression of MYCN rescued the decreased malignant phenotype caused by over-expression of miR-98 in NB. N6-methyladenosine modification in 3′-UTR of MYCN promoted its interaction with miR-98. The data collectively demonstrated that RNA m6A modification was required for miR-98/MYCN axis-mediated inhibition of neuroblastoma progression, and miR-98 might be novel targets for NB detection and treatment.

The Role of Adjuvant Treatment in Craniofacial Malignancy: A Critical Review.

Background: Tumors originating from the craniofacial region usually present in a locally advanced stage with frequent involvement of adjacent sites and have a strong tendency for local recurrence in the absence of adjuvant therapy, even when the original surgical resection was presumed to be radical. In the past decades, several advances in the radiological diagnosis and treatment of craniofacial malignancies have been introduced. There are, however, no randomized trials that define the optimal multimodal treatment of these tumors because of their rarity as well as heterogeneity in both histology and site of origin. The aim of this study was to conduct a critical review of the role of adjuvant therapy in the treatment of craniofacial malignancy.Method: We conducted a critical review of the past and contemporary literature available, focusing on adjuvant oncological treatments of the most common craniofacial malignancies.Results: Preoperative radiotherapy can have a documented role in the treatment of olfactory neuroblastoma and soft tissue sarcoma, while preoperative chemotherapy can be advocated in the treatment of sinonasal undifferentiated carcinoma, neuroendocrine carcinoma, olfactory neuroblastoma, and craniofacial sarcoma (both soft-tissue and high-grade osteosarcoma). Postoperative radiotherapy has a well-established role in the treatment of most craniofacial malignancies. The role of postoperative chemotherapy is unclear in most histologies, but is commonly used during the treatment of well-selected cases of paranasal sinus carcinoma, olfactory neuroblastoma, mucosal melanoma, soft tissue sarcoma and high-grade craniofacial osteosarcoma.Discussion: Alongside developments in surgery, there have also been improvements in diagnostics, radiotherapy, and chemotherapy. Implementation of novel radiation techniques allows delivery of higher radiation doses while minimizing irradiation-related morbidity. Better understanding of tumor biology allows the construction of more complex treatment strategies, incorporating adjuvant chemotherapy either pre- or postoperatively. In the era of personalized targeted therapy, rapid strides are being made to identify specific tumor-targets for use of novel biologic agents, with the potential to change current management paradigms.

<p>LncRNA DLX6-AS1 Promotes the Progression of Neuroblastoma by Activating STAT2 via Targeting miR-506-3p</p>

BackgroundNeuroblastoma (NB) is a common malignant tumor of the sympathetic nervous system, mainly disturbing children. Long non-coding RNAs (lncRNAs) serving as promising cancer biomarkers have been well recognized. Our study intends to explore the functions of lncRNA X–inactive specific transcript (DLX6-AS1) in NB and provide a potential action mechanism.MethodsThe expression of DLX6-AS1, miR-506-3p and signal transducer and activator of transcription 2 (STAT2) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay. Cell cycle distribution was determined by flow cytometry assay. The protein level of cell cycle-related markers and STAT2 was detected by Western blot. Glycolysis progress was evaluated according to glucose consumption, lactate production and ATP level. The target genes were predicted by the online database Starbase3.0 and verified by dual-luciferase reporter assay.ResultsDLX6-AS1 expression was highly elevated in NB tissues and cells. DLX6-AS1 deficiency inhibited NB cell proliferation, cell cycle and glycolysis in vitro. MiR-506-3p was a target of DLX6-AS1, and miR-506-3p absence partly reversed the effects of DLX6-AS1 deficiency. Besides, STAT2 was targeted by miR-506-3p, and its expression was regulated by DLX6-AS1 through miR-506-3p. MiR-506-3p restoration also inhibited NB cell malignant behaviors, and STAT2 overexpression partially abolished the role of miR-506-3p restoration. Moreover, DLX6-AS1 deficiency weakened tumor growth in vivo.ConclusionDLX6-AS1 regulated cell proliferation, cell cycle and glycolysis in vitro and tumor growth in vivo to promote the development of NB by upregulating STAT2 via targeting miR-506-3p.

Circular RNA DGKB Promotes the Progression of Neuroblastoma by Targeting miR-873/GLI1 Axis.

Accumulated evidences suggested that circular RNAs (circRNA) played critical roles in tumorigenesis and progression. To our knowledge, no study reported the function of circular RNA DGKB (circDGKB, circRNA ID: hsa_circ_0133622) on progression of neuroblastoma (NB). Here, we showed that circDGKB was upregulated in NB tissues compared to the normal dorsal root ganglia. Moreover, the expression level of circDGKB was negatively correlated with the survival rate of NB patients. Mechanically, overexpression of circDGKB promoted the proliferation, migration, invasion, and tumorigenesis of NB cells and reduced cell apoptosis, and vice versa. In addition, qRT-PCR and/or Western blot results showed that circDGKB overexpression inhibited the expression level of miR-873 and enhanced GLI1 expression. Moreover, miR-873 functioned an opposite role to circDGKB and significantly weakened circDGKB role in promoting NB progression. Furthermore, GLI1 upregulation also rescued the miR-873 role in inhibiting NB progression. In conclusion, our work proved that circDGKB promoted NB progression via targeting miR-873/GLI1 axis in vitro and in vivo. Our study provided a new target for NB treatment and indicated that circDGKB could act as a novel diagnostic marker for NB.

Abstract PR03: Sex ratio disparities and the risk of childhood cancer: Evaluating the mediating effect of birth defects among 15,000 childhood cancer cases

Abstract Background: There is a persistent yet unexplained disparity in the sex ratio among childhood cancer cases, whereby males are more likely to develop several cancers. These patterns are also seen for most birth defects, which are strongly associated with the risk of childhood cancer. We conducted a mediation analysis to estimate whether birth defect status mediates the association between sex and childhood cancer risk. Methods: We linked data from birth, birth defects, and cancer registries to obtain a population-based birth cohort including births from 1992-2013 in Arkansas, Michigan, North Carolina, and Texas. We obtained birth defects diagnoses, including chromosomal and nonchromosomal defects, from statewide, population-based birth defects registries. We focused on “major” defects as defined by the National Birth Defects Prevention Network and the National Birth Defects Prevention Study. Cancer diagnoses that occurred prior to age 18 years were obtained from state cancer registries. We obtained data on child’s sex, birth weight, gestational age, maternal race/ethnicity, maternal age, and plurality from birth records. We conducted counterfactual mediation analysis to estimate the direct and indirect effects of sex on risk of childhood cancer, modeling sex as the exposure, birth defect status as the mediator, and cancer type as the outcome. State, maternal race/ethnicity (white, black, other), maternal age (continuous), and plurality (singleton vs. multiple) were considered potential mediator-outcome confounders. Due to lower success rates of linkage to birth records among adolescent cancer cases, we conducted a sensitivity analysis limiting to cancers diagnosed at age < 5 years. Results: Our dataset included 10,181,074 children (5,208,379 males; 4,972,695 females), including 15,110 with cancer diagnoses (8044 males; 7066 females), 539,567 children with birth defects diagnoses (320,666 males; 218,901 females), and 2,124 co-occurring cases (children with both cancer and one or more birth defect diagnosis: 1,186 males; 938 females). We observed mediation of the association between sex and childhood cancer overall by birth defect status (proportion mediated [PM]: 38%). In analyses of specific cancer types, we observed variation of the estimated proportion mediated among several cancers, including hepatoblastoma (PM: 33%), neuroblastoma (PM: 35%), non-Hodgkin lymphoma (PM: 6%), and soft tissue sarcomas (PM: 25%). In analysis of children age < 5 years at cancer diagnosis, we observed similar results. Conclusions: Our results suggest that birth defects mediate a significant proportion of the overall relationship between sex and childhood cancer. While approximately 60% of the male excess in childhood cancer cases remains unexplained, these findings add to our understanding of the causal pathway of male sex as a risk factor for childhood cancer. Additional studies are under way to characterize the biology underlying these observations. This abstract is also being presented as Poster A66. Citation Format: Erin L. Marcotte, Jeremy M. Schraw, Tania A. Desrosiers, Wendy N. Nembhard, Peter H. Langlois, Mark A. Canfield, Robert E. Meyer, Sharon E. Plon, Philip Lupo. Sex ratio disparities and the risk of childhood cancer: Evaluating the mediating effect of birth defects among 15,000 childhood cancer cases [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR03.

LncRNA NORAD accelerates the progression and doxorubicin resistance of neuroblastoma through up-regulating HDAC8 via sponging miR-144-3p

The dysregulation of non-coding RNAs (ncRNAs) often caused aberrant cell behaviors. In the present study, we focused on the role of long noncoding RNA (lncRNA) non-coding RNA activated by DNA damage (NORAD) in the development of neuroblastoma (NB). The enrichment of NORAD, miRNA-144-3p (miR-144-3p) and histone deacetylase 8 (HDAC8) was measured by quantitative real time polymerase chain reaction (qRT-PCR). The proliferation, chemoresistance, apoptosis, metastasis and autophagy of NB cells were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, transwell migration and invasion assays and Western blot assay, respectively. The target relationship between miR-144-3p and NORAD or HDAC8 was predicted by Starbase software and validated through dual-luciferase reporter assay, RIP and RNA-pull down assays. The protein expression of HDAC8 was measured by Western blot assay. Murine xenograft model was used to verify the function of NORAD in vivo. We found that the level of NORAD was up-regulated in NB tissues and cells, and the level of NORAD was negatively correlated with the prognosis of NB patients. NORAD promoted the proliferation, metastasis and doxorubicin (DOX) resistance while inhibited the apoptosis and autophagy of NB cells. MiR-144-3p was a target of NORAD in NB cells, and NORAD accelerated the progression and DOX resistance of NB through sponging miR-144-3p. HDAC8 was a direct target of miR-144-3p in NB cells, and miR-144-3p suppressed the progression of NB through down-regulating HDAC8. NORAD up-regulated the expression of HDAC8 through sponging miR-144-3p in NB cells. NORAD accelerated the growth of NB tumors at least partly through miR-144-3p/HDAC8 signaling in vivo. In conclusion,NORAD promoted the progression and DOX resistance of NB through miR-144-3p/HDAC8 axis in vivo and in vitro.

SNHG16 promotes tumorigenesis and cisplatin resistance by regulating miR-338-3p/PLK4 pathway in neuroblastoma cells

BackgroundLong noncoding RNA small nucleolar RNA host gene 16 (lncRNA SNHG16) has been revealed to be involved in the tumorigenesis of neuroblastoma. However, the role of SNHG16 in regulating cisplatin sensitivity in neuroblastoma remains largely unknown.MethodsThe expression of SNHG16, microRNA (miR)-338-3p and polo-like kinase 4 (PLK4) mRNA was measured using quantitative real-time polymerase chain reaction. The protein levels of PLK4, multidrug resistance protein 1 (MRP1), multidrug-resistance gene 1-type p-glycoprotein (P-gp) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins were detected by Western blot. The half maximal inhibitory concentration (IC50) value, cell proliferation, migration and invasion were analyzed using Cell Counting Kit-8 assays or Transwell assay. Apoptotic cells were measured by Flow cytometry. The interaction between miR-338-3p and SNHG16 or PLK4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assay. In vivo experiments were conducted through the murine xenograft model.ResultsSNHG16 was up-regulated, while miR-338-3p was down-regulated in cisplatin-resistant neuroblastoma tissues and cells. SNHG16 silencing weakened cisplatin resistance, reflected by the reduction of IC50 value, down-regulation of MRP-1 and P-gp protein expression, suppression of proliferation, migration and invasion, as well as enhancement of apoptosis in SNHG16 deletion cisplatin-resistant neuroblastoma cells. Besides that, SNHG16 could regulate PLK4 expression by sponging miR-338-3p and SNHG16/miR-338-3p/PLK4 axis could affect the activation of PI3K/AKT pathway in cisplatin-resistant neuroblastoma cells. MiR-338-3p inhibition attenuated SNHG16 deletion-mediated impairment on cisplatin resistance and PLK4 overexpression reversed the decrease of cisplatin-resistance induced by miR-338-3p re-expression. Furthermore, SNHG16 knockdown contributed to the anti-tumor effect of cisplatin in neuroblastoma in vivo.ConclusionSNHG16 contributed to the tumorigenesis and cisplatin resistance in neuroblastoma possibly through miR-338-3p/PLK4 pathway, indicating a novel insight for overcoming chemoresistance in neuroblastoma patients.

Long non-coding RNA SNHG7 promotes neuroblastoma progression through sponging miR-323a-5p and miR-342-5p

Dysregulation of long non-coding RNAs (lncRNAs) has been known to be relevant to the progression of human cancers, including neuroblastoma (NB). Small nucleolar RNA host gene 7 (SNHG7) has been identified as an oncogene in a series of human cancers. The purpose of the present study was to investigate the function and underlying mechanism of SNHG7 in NB progression. qRT-PCR was used to determine the levels of SNHG7, cyclin D1 (CCND1), miR-323a-5p and miR-342-5p. Cell migration and invasion abilities were detected by transwell assays. Glucose consumption and lactate production were assessed using the corresponding assay kits. The targeted interaction between SNHG7 and miR-323a-5p or miR-342-5p was verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Xenograft tumor assays were performed to observe the effect of SNHG7 silencing on tumor growth in vivo. We found that SNHG7 was upregulated in NB tissues and cell lines, and high SNHG7 level was relevant to poor prognosis of NB patients. SNHG7 silencing resulted in the repression of NB cell migration, invasion and glycolysis. SNHG7 directly targeted miR-323a-5p and miR-342-5p and negatively modulated their expression in NB cells. The overexpression of miR-323a-5p or miR-342-5p weakened NB cell migration, invasion and glycolysis. Moreover, miR-323a-5p or miR-342-5p mediated the suppressive effect of SNHG7 silencing on NB cell progression. CCND1 was a direct target of miR-323a-5p and miR-342-5p. Additionally, SNHG7 knockdown repressed tumor growth in vivo. In conclusion, our study suggested that SNHG7 silencing hindered NB progression at least partly though sponging miR-323a-5p and miR-342-5p, illuminating its potential value as a therapeutic target.

MiR-424 suppressed viability and invasion by targeting to the DCLK1 in neuroblastoma.

Neuroblastoma is the most frequent tumor of sympathetic nervous system in infants. MiRNAs acted as oncogenes or tumor suppressors in the process of tumor development. We aim at exploring the functions of miRNA in neuroblastoma. Cell viability and invasion were evaluated by Cell Counting Kit-8 (CCK-8) and transwell assays. Western blot was utilized to assess the protein expression associated with epithelial-mesenchymal transition (EMT) markers. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to calculate the mRNA levels of miRNA and gene. MiR-424 was downregulated while doublecortin like kinase 1 (DCLK1) was upregulated in neuroblastoma tissues and cells compared to adjacent non-tumor and normal spongiocyte cells. MiR-424 suppressed cell viability, invasion, and EMT by targeting DCLK1. MiR-424 regulated the expression of DCLK1 by directly binding to the 3'-untranslated region (UTR) of DCLK1 mRNA in SK-N-SH and Be2C cells. DCLK1 reversed partial functions of miR-424 in neuroblastoma. MiR-424 suppressed cell viability, invasion, and EMT by directly targeting the 3'-UTR of DCLK1 mRNA. The newly identified miR-424/DCLK1 axis provides novel insights into the pathogenesis of neuroblastoma.

MiR-542-3p Suppresses Neuroblastoma Cell Proliferation and Invasion by Downregulation of KDM1A and ZNF346.

Neuroblastoma is one of the most common malignancies in infants and children. MicroRNAs (miRNAs) have been reported as significant regulators that play important roles in neuroblastoma development. This research aimed to analyze the functional mechanism of miR-542-3p in neuroblastoma. Here, we found that miR-542-3p was downregulated and KDM1A as well as ZNF346 were upregulated in neuroblastoma tissues and cells. Both overexpression of miR-542-3p and the knockdown of KDM1A suppressed cell proliferation and invasion in neuroblastomas. Moreover, miR-542-3p reduced the levels of KDM1A and ZNF346 through interaction. Both KDM1A overexpression and ZNF346 upregulation weakened the effect of miR-542-3p on neuroblastoma cells. Besides, miR-542-3p negatively regulated tumor growth in vivo. Our results suggested that miR-542-3p suppressed cell proliferation and invasion by targeting KDM1A and ZNF346 in neuroblastomas, providing a theoretical basis for the treatment of neuroblastoma.

Investigation of the Oncogenic Role of the Cell Cycle Regulator SAPCD2 in Pediatric Neuroblastoma

Pediatric neuroblastoma is the most common extra‐cranial malignancy in infants and children. Neuroblastoma is a neuroendocrine cancer arising from sympathetic neuronal precursors that circumvent the differentiation process into mature neurons and glia. The poor differentiation status of neuroblastoma cells allows them to retain features of early embryonic stem cells. SAPCD2 is a newly identified cell cycle regulator involved in mitotic progression and spindle assembly. Although the mechanism by which SAPCD2 exerts its effect is poorly understood, its function appears to be critical in early embryonic development where it regulates mitotic spindle orientation, planar polarization in cell progenitors, and tissue segmentation. More relevant to this study, a recent investigation has revealed that SAPCD2 plays a role in symmetric planar divisions during early embryonic segmentation by interacting with the Gαi‐LGN‐NuMA ternary spindle assembly complex, which appears to be critically important for modulating differentiation of neuroepithelial cells. The expression of SAPCD2 is generally exclusive to embryonic tissues except in the case of malignancies where it is dramatically overexpressed compared to normal tissues. Its overexpression has been observed in several cancers such as in the lung and liver, but its relevance to neuroblastoma has not been reported in the current literature. The objective of this study is to investigate whether SAPCD2 expression levels in human neuroblastoma tissues are correlated with disease progression and if so, to further characterize its role in regulating neuroblastoma cell survival in vitro. The correlation of SAPCD2 with neuroblastoma patient survival was investigated using two data sets obtained from the public R2: Genomics database ( http://r2.amc.nl). The patients were divided into two groups based on SAPCD2 mRNA level (high or low), and the difference in patient survival between the two groups was compared using Kaplan‐Meier analysis. To further investigate the potential oncogenic role of SAPCD2 in vitro, the neuroblastoma cell line BE(2)‐C was transfected with siRNAs against SAPCD2 mRNA to determine the effect of SAPCD2 knockdown on neuroblastoma cell survival and proliferation. Our results demonstrate a statistically significant difference in the survival probabilities of neuroblastoma patients within the two SAPCD2 expression groups, with patients possessing high SAPCD2 mRNA levels showing significantly reduced survival compared to patients with low SAPCD2 mRNA level in both datasets. In in vitro experimental investigations, siRNA‐mediated knockdown of SAPCD2 caused a dramatic reduction in neuroblastoma cell viability after five days and a dramatic reduction in colony formation capacity after 14 days. These results strongly suggest that SAPCD2 functions as an oncogene in neuroblastoma and elevated SAPCD2 expression in neuroblastoma cells promote poor patient prognosis. Future elucidation of the role of SAPCD2 in contributing to the oncogenic transformation of neuroblastoma may present a novel target for drug development which may enhance therapies for neuroblastoma.Support or Funding InformationTexas State University, National Institutes of Health

LncRNA cancer susceptibility candidate (CASC7) upregulates phosphatase and tensin homolog by downregulating miR-10a to inhibit neuroblastoma cell proliferation.

Long non-coding (lncRNA) cancer susceptibility candidate (CASC7) plays a tumor-suppressive role in several malignancies. In this study, the role of CASC7 in neuroblastoma was investigated for the first time. We observed the downregulation of CASC7 in neuroblastoma tissues compared to non-cancer tissues of neuroblastoma patients. Across neuroblastoma tissues, CASC7 was inversely correlated with microRNA-10a (miR-10a) but positively correlated with phosphatase and tensin homolog mRNA. In neuroblastoma cells, CASC7 overexpression led to downregulated miR-10a but upregulated phosphatase and tensin homolog. Furthermore, miR-10a overexpression led to downregulated phosphatase and tensin homolog and reduced effects of CASC7 overexpression. CASC7 overexpression resulted in inhibition, while miR-10a overexpression resulted in increased proliferation rate of neuroblastoma cells. We therefore concluded that lncRNA CASC7 may upregulate phosphatase and tensin homolog by downregulating miR-10a to inhibit neuroblastoma cell proliferation.

Ubiquitin-specific protease 22 is associated with poor prognosis in neuroblastoma.

Ubiquitin-specific protease 22 (USP22) alters histone ubiquitination and is considered to be an oncogenic factor involved in tumor progression. The USP22 aberrance has been implicated in several malignancies, but whether USP22 plays a role in neuroblastoma (NB) remains unclear. To the best of our knowledge, the clinicopathological significance of USP22 expression in NB has not been previously reported in the English-language medical literature. The aim of this study was to investigate the role of USP22 and its association with potential targets in patients with NB. The potential clinicopathological significance of USP22 expression in NB was studied using immunohistochemistry, immunohistochemical staining assessment and statistical analyses. Based on the immunohistochemical analysis, the USP22 protein was detected more manifestly in NB tissues than in healthy peritumoral tissue. Furthermore, an association between USP22, lymph node metastasis and NB clinical stage was observed, whereby the level of USP22 protein was higher in stage III-IV specimens than in stage I-II specimens (p < 0.05). Furthermore, tumors expressing USP22 were associated with poorer patient prognosis than the USP22-negative tumors. The multivariate Cox regression analysis suggested that the level of USP22 protein is a predictive factor for survival (p < 0.05). Our results indicate a significant association between USP22 level and poor prognosis in NB. Thus, USP22 represents a valuable biomarker for predicting the outcome of patients with NB.

SNHG16 Silencing Inhibits Neuroblastoma Progression by Downregulating HOXA7 via Sponging miR-128-3p.

Neuroblastoma (NB) is a common intracranial solid tumor with high mortality. Small nucleolar RNA host gene 16 (SNHG16), one of the long noncoding RNAs (lncRNAs), has been reported to be linked to the poor prognosis of NB. However, the mechanisms of SNHG16 in regulating NB progression remain poorly understood. The expression level of SNHG16 was measured by quantitative real time polymerase chain reaction (qRT-PCR). The starBase was employed to predict the interaction of miR-128-3p and SNHG16 or HOXA7, which was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell proliferation and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. Transwell assay was used to detect cell invasion or migration. The mRNA and protein levels of homeobox protein A7 (HOXA7) were determined by qRT-PCR and western blot, respectively. The levels of SNHG16 and HOXA7 were conspicuously increased in NB tissues and cells, while the expression of miR-128-3p was obviously declined, compared with corresponding normal tissues and cells. SNHG16 silencing inhibited proliferation, migration and invasion and induced apoptosis of NB cells. We identified that SNHG16 directly interacted with miR-128-3p, and miR-128-3p could target the 3'UTR of HOXA7 in NB cells. Simultaneously, miR-128-3p expression was negatively associated with SNHG16 or HOXA7. Further studies indicated that SNHG16 overexpression rescued the effects of miR-128-3p-mediated on inhibiting proliferation, migration, invasion and promoting apoptosis of NB cells. Moreover, SNHG16 could modulate HOXA7 by sponging miR-128-3p in NB cells. Besides, SNHG16 silencing suppressed tumor growth in vivo. Knockdown of SNHG16 impeded proliferation, migration, invasion and induced apoptosis through the SNHG16/miR-128-3p/HOXA7 axis in NB cells.

LncRNA SNHG4 promotes neuroblastoma proliferation, migration, and invasion by sponging miR-377-3p.

Long non-coding RNAs (lncRNAs) have been demonstrated to act as essential regulators in the growth and progression of neuroblastoma. In the present research, the high expression of lncRNA small nucleolar RNA host gene 4 (SNHG4) in neuroblastoma was tested via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and then the function of SNHG4 was explored and verified by CCK-8 assay, EdU assay, cell cycle assay, cell apoptosis test, wound healing test and invasion test in neuroblastoma cell lines. It was discovered that lncRNA SNHG4 exhibited high expression in neuroblastoma tissues and cell lines, and the expression of SNHG4 was associated with the survival of neuroblastoma patients. Additionally, SNHG4 decrement markedly repressed neuroblastoma cells to proliferate and stimulate their apoptosis in vivo and in vitro. Moreover, SNHG4 decrement impeded the abilities of SH-SY5Y and IMR-32 cells to migrate and invade as well as epithelial-mesenchymal transition (EMT). In mechanism, we found that SNHG4 acted as a competing endogenous RNA to sponge miR-377-3p, which was downregulated in neuroblastomas and inhibited cell proliferation and invasion. The findings manifested that SNHG4 was inversely associated with miR-377-3p expression in neuroblastoma cases. Collectively, we revealed the functions of SNHG4 and miR-377-3p in neuroblastoma.

MiR-490-5p functions as tumor suppressor in childhood neuroblastoma by targeting MYEOV.

Neuroblastoma (NB) is the most common extracranial solid tumor derived from neural crest in children. Recently, the role of miRNA has been studied extensively in the development of NB. Here, we investigated the clinical significance of microRNA-490-5p (miR-490-5p) in NB. A total of 72 pairs of NB tumor tissues and matched adjacent normal nerve tissues were collected from NB patients. The expression of miR-490-5p was significantly down-regulated in NB tissues and cell lines using quantitative real-time PCR. Using Pearson Chi-square test and Kaplan-Meier analysis, we found that significantly decreased miR-490-5p levels were correlated with INSS stage, lymph-node metastasis, and poor survival prognosis in NB patients. MiR-490-5p overexpression significantly suppressed cell proliferation migration, invasion, and induced cell cycle G0/G1 arrest and cell apoptosis in NB cell lines (SH-SY5Y and SK-N-SH) using CCK-8, flow cytometry, and transwell assays. Mechanistically, MYEOV was confirmed as a target gene of miR-490-5p by luciferase reporter assay. Furthermore, MYEOV knockdown imitated, while overexpression rescued the changes in the biological features of miR-490-5p on NB cells. Our results demonstrated for the first time that miR-490-5p functions as a tumor suppressor in NB by targeting MYEOV, which might provide novel approaches for the treatment of NB.

NEAT1 Negatively Regulates Cell Proliferation and Migration of Neuroblastoma Cells by miR-183-5p/FOXP1 Via the ERK/AKT Pathway

Neuroblastoma, a malignant tumor of the sympathetic nervous system, is an aggressive extracranial tumor in childhood. Long noncoding RNAs (lncRNAs) have been discovered to play a key role in the eukaryotic regulatory gene network and be involved in a wide variety of biological processes. We observed that the expression of lncRNA nuclear-enriched abundant transcript-1 (NEAT1) was significantly decreased in human neuroblastoma tissues and cell lines, compared with the normal. We observed cell proliferation, migration, and invasion with Cell Counting Kit-8 assay, colony formation assay, and Transwell assay to investigate the effects of NEAT1, miR-183-5p, or FOXP1 on neuroblastoma cells. And we also used StarBase and luciferase reporter gene assay to predict and confirm the interaction of NEAT1, miR-183-5p, and FOXP1 in neuroblastoma cells. First, overexpression of NEAT1 suppressed cell proliferation and played a key role in cell migration and invasion. In addition, NEAT1 was demonstrated to directly interact with miR-183-5p and exerted its antioncogenic role in neuroblastoma by negatively regulating miR-183-5p expression. miR-183-5p suppressed the expression of FOXP1 and regulated cell proliferation and migration by directly targeting FOXP1 mRNA 3′-untranslated region. Moreover, FOXP1 antagonized the effect of miR-183-5p on the phosphorylation of extracellular-regulated kinase/protein kinase B (ERK/AKT), while FOXP1 siRNA increased the reduced phosphorylation of ERK/AKT caused by miR-183-5p inhibitor in neuroblastoma cells. Taken together, these data showed that NEAT1 negatively regulated cell proliferation and migration of neuroblastoma by the miR-183-5p/FOXP1 axis via suppression of the ERK/AKT pathway. Our findings may provide a new target for the study of pathogenesis and treatment of neuroblastoma.

CHL1 and NrCAM are Primarily Expressed in Low Grade Pediatric Neuroblastoma.

BackgroundNeural cell adhesion molecules like close homolog of L1 protein (CHL1) and neuronal glia related cell adhesion molecule (NrCAM) play an important role in development and regeneration of the central nervous system. However, they are also associated with cancerogenesis and progression in adult malignancies, thus gain increasing importance in cancer research. We therefore studied the expression of CHL1 and NrCAM according to the course of disease in children with neuroblastoma.MethodsCHL1 and NrCAM expression levels were histologically assessed by tissue microarrays from surgically resected neuroblastoma specimens of 56 children. Expression of both markers was correlated to demographics as well as clinical data including metastatic dissemination and survival.ResultsCHL1 was expressed in 9% and NrCAM in 51% of neuroblastoma tissue samples. Expression of CHL1 was higher in patients with low Hughes grade 1a/b (p=0.01). NrCAM was more often detected in patients with a low International Staging System (INSS) score 1/2 (p=0.04).ConclusionCHL1 and NrCAM expression was associated with low-grade pediatric neuroblastoma. These adhesion molecules may play a role in early tumor development of neuroblastoma.