Neuroblastoma Cell Proliferation Research Articles

LINC00839 Regulates Proliferation, Migration, Invasion, Apoptosis and Glycolysis in Neuroblastoma Cells Through miR-338-3p/GLUT1 Axis.

BackgroundLong noncoding RNAs (lncRNAs) are related to the development and treatment of neuroblastoma. The lncRNA LINC00839 is dysregulated in neuroblastoma, while its function and mechanism in neuroblastoma development remain largely unclear.MethodsThe tumor and adjacent noncancerous tissues were collected from 48 neuroblastoma patients. LINC00839, glucose transporter 1 (GLUT1) and microRNA-338-3p (miR-338-3p) abundances were examined via quantitative reverse transcription polymerase chain reaction or Western blot. Cell proliferation, apoptosis, migration, invasion and glycolysis were assessed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing, transwell, glucose uptake and lactate production. The target relationship of miR-338-3p and LINC00839 or GLUT1 was tested via dual-luciferase reporter analysis and RNA immunoprecipitation. The function of LINC00839 on neuroblastoma cell growth in vivo was tested via a xenograft model.ResultsLINC00839 and GLUT1 abundances were increased in neuroblastoma tissues and cell lines. The high expression of LINC00839 and GLUT1 indicated the lower overall survival. LINC00839 interference constrained neuroblastoma cell proliferation, migration, invasion and glycolysis, and facilitated apoptosis. GLUT1 overexpression or miR-338-3p knockdown could mitigate the influence of LINC00839 silence on neuroblastoma cell processes. LINC00839 could regulate GLUT1 expression via miR-338-3p. LINC00839 knockdown reduced neuroblastoma cell growth in xenograft model.ConclusionLINC00839 silence repressed neuroblastoma cell proliferation, migration, invasion and glycolysis and promoted apoptosis via regulating miR-338-3p/GLUT1 axis.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA promotes the proliferation, invasion and migration of neuroblastoma cells via targeting β-1, 4-galactosyltransferase III by sponging miR-338-3p.

Neuroblastoma is a common malignant tumor in children, and patients often have a poor prognosis. Long noncoding RNAs (lncRNAs) are involved in the regulation of neuroblastoma progression. However, the regulatory effect of lncRNA differentiation antagonizing nonprotein coding RNA (DANCR) on neuroblastoma is still not clear. The expression levels of DANCR, miR-338-3p and β-1, 4-galactosyltransferase III (B4GALT3) were determined by quantitative real-time PCR. 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, flow cytometry and transwell assays were used to evaluate the proliferation, apoptosis, migration and invasion abilities of neuroblastoma cells. Moreover, western blot analysis was performed to assess the levels of B4GALT3 and the proliferation, apoptosis and migration-related proteins. Besides, a dual-luciferase reporter assay was used to verify the interactions among DANCR, miR-338-3p and B4GALT3. Mice xenograft models were used to ascertain the effect of DANCR on neuroblastoma tumor growth in vivo. Our results revealed that DANCR was highly expressed in neuroblastoma tissues and cells, and its silencing impeded the progression of neuroblastoma cells. DANCR could interact with miR-338-3p. Knockdown of miR-338-3p recovered the inhibitory effect of DANCR knockdown on neuroblastoma progression. B4GALT3 was a target of miR-338-3p. B4GALT3 overexpression reversed the suppression effect of DANCR silencing on neuroblastoma progression. In-vivo experiments further confirmed that DANCR silencing inhibited neuroblastoma tumor growth. Our results indicated that DANCR promoted B4GALT3 expression to increase the proliferation, migration and invasion of neuroblastoma cells via sponging miR-338-3p, which provided a theoretical basis for the targeted therapy of neuroblastoma.

MiRNA-34a-5p regulates progression of neuroblastoma via modulating the Wnt/β-catenin signaling pathway by targeting SOX4.

Neuroblastoma is an embryonal tumor of the autonomic nervous system with poor prognosis in children. In present study, we demonstrated the relationship of miRNA-34a-5p in the regulating of the Wnt/β-catenin signaling pathway by targeting SRY-related HMG-box (SOX4)Reverse transcription-quantitative PCR was used to detect the expression levels of miRNA-34a-5p and SoX4. Western blotting was performed to assess the protein expression levels of SoX4, Wnt, MMP9, Bax, and Bcl-2. The proliferation, apoptosis, migration and invasion of neuroblastoma cells were determined using MTT, flow cytometry and Transwell assays.In this study, we sought to investigate the role of miRNA-34a-5p on neuroblastoma and the possible molecular mechanism. We had performed in-vitro and in-vivo experiments to evaluate the effects of miRNA-34a-5p on neuroblastoma cell proliferation and invasion by altering its expression level via cell transfection. On the basis of our study, miRNA-34a-5p showed decreased expression levels in neuroblastoma. Subsequently, we manipulated miRNA-34a-5p expression through cell transfection and observed abnormal expression of β-catenin as well as the downstream targets of the Wnt/β-catenin pathway in neuroblastoma cells. With all these evidences, we determined that miRNA-34a-5p regulated Wnt/β-catenin pathway by targeting SOX4.In conclusion, our study demonstrates that miRNA-34a-5p can inhibit the over-activation of the Wnt/β-catenin signaling pathway via targeting SOX4 and further regulate proliferation, invasion of neuroblastoma cells.

Ubiquitination of p21 by E3 Ligase TRIM21 Promotes the Proliferation of Human Neuroblastoma Cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, which shows great clinical and biomolecule heterogeneity. Currently, surgery is still the main method of neuroblastoma treatment and specific therapeutic drugs are lacking, so useful targets are urgently needed. TRIM21 is a RING-type E3 ligase that its overexpression promotes the progression of human glioma, while whose effects on neuroblastoma have not been illustrated. Firstly, the shRNAs targeting TRIM21 were designed and found that the ablation of TRIM21 inhibits the proliferation of human neuroblastoma cells. Then the molecular mechanism study indicated that TRIM21 interacts with, and mediates p21 degradation by ubiquitination modification. Further study demonstrates that TRIM21 regulates the proliferation of neuroblastoma cells in a p21-dependent manner. These results suggest that TRIM21 might be a potential therapeutic target for neuroblastoma.

Activation of Cilia-Independent Hedgehog/GLI1 Signaling as a Novel Concept for Neuroblastoma Therapy

Simple SummaryElevated GLI1 expression levels are associated with improved survival in NB patients and GLI1 overexpression exerts tumor-suppressive traits in cultured NB cells. However, NB cells are protected from increased GLI1 levels as they have lost the ability to form primary cilia and transduce Hedgehog signals. This study identifies an isoxazole (ISX) molecule with primary cilia-independent GLI1-activating properties, which blocks NB cell growth. Mechanistically, ISX combines the removal of GLI3 repressor and the inhibition of class I HDACs, providing proof-of-principle evidence that small molecule-mediated activation of GLI1 could be harnessed therapeutically in the future.Although being rare in absolute numbers, neuroblastoma (NB) represents the most frequent solid tumor in infants and young children. Therapy options and prognosis are comparably good for NB patients except for the high risk stage 4 class. Particularly in adolescent patients with certain genetic alterations, 5-year survival rates can drop below 30%, necessitating the development of novel therapy approaches. The developmentally important Hedgehog (Hh) pathway is involved in neural crest differentiation, the cell type being causal in the etiology of NB. However, and in contrast to its function in some other cancer types, Hedgehog signaling and its transcription factor GLI1 exert tumor-suppressive functions in NB, rendering GLI1 an interesting new candidate for anti-NB therapy. Unfortunately, the therapeutic concept of pharmacological Hh/GLI1 pathway activation is difficult to implement as NB cells have lost primary cilia, essential organelles for Hh perception and activation. In order to bypass this bottleneck, we have identified a GLI1-activating small molecule which stimulates endogenous GLI1 production without the need for upstream Hh pathway elements such as Smoothened or primary cilia. This isoxazole compound potently abrogates NB cell proliferation and might serve as a starting point for the development of a novel class of NB-suppressive molecules.

FABP4 deactivates NF-κB-IL1α pathway by ubiquitinating ATPB in tumor-associated macrophages and promotes neuroblastoma progression.

Neuroblastoma (NB) is the most common and deadliest pediatric solid tumor. Targeting and reactivating tumor‐associated macrophages (TAMs) is necessary for reversing immune suppressive state and stimulating immune defense to exert tumoricidal function. However, studies on the function and regulation of TAMs in NB progression are still limited. Fatty acid binding protein 4 (FABP4) in TAMs was correlated with advanced clinical stages and unfavorable histology of NB. FABP4‐mediated macrophages increased migration, invasion, and tumor growth of NB cells. Mechanically, FABP4 could directly bind to ATPB to accelerate ATPB ubiquitination in macrophages. The consequently decreased ATP levels could deactivate NF‐κB/RelA‐IL1α pathway, which subsequently results in macrophages reprogrammed to an anti‐inflammatory phenotype. We also demonstrated that FABP4‐enhanced migration and invasion were significantly suppressed by IL1α blocking antibody. Furthermore, circulating FABP4 was also associated with the clinical stages of NB. Our findings suggest that FABP4‐mediated macrophages may promote proliferation and migration phenotypes in NB cells through deactivating NF‐κB‐IL1α pathway by ubiquitinating ATPB. This study reveals the pathologic and biologic role of FABP4‐mediated macrophages in NB development and exhibits a novel application of targeting FABP4 in macrophages for NB treatment.

The loss of DLG2 isoform 7/8, but not isoform 2, is critical in advanced staged neuroblastoma

BackgroundNeuroblastoma is a childhood neural crest tumor showing large clinical and genetic heterogeneity, one form displaying 11q-deletion is very aggressive. It has been shown that 11q-deletion results in decreased expression of DLG2, a gene residing in the deleted region. DLG2 has a number of different isoforms with the main difference is the presence or absence of a L27 domain. The L27 domain containing DLG proteins can form complexes with CASK/MPP and LIN7 protein family members, which will control cell polarity and signaling.MethodsWe evaluated the DLG gene family and the LIN7 gene family for their expression in differently INSS staged neuroblastoma from publically available data and primary tumors, we included two distinct DLG1 and DLG2 N-terminal transcript isoforms encoding L27 domains for their expression. Functionality of DLG2 isoforms and of LIN7A were evaluated in the 11q-deleted neuroblastoma cell line SKNAS.ResultsIn neuroblastoma only two DLG2 isoforms were expressed: isoform 2 and isoform 7/8. Using the array data we could determine that higher expression of DLG members that contain L27 domains correlated to better survival and prognosis. Whilst DLG1 showed a decrease in both isoforms with increased INSS stage, only the full length L27 containing DLG2 transcripts DLG2-isoform 7/8 showed a decrease in expression in high stage neuroblastoma. We could show that the protein encoded by DLG2-isoform 7 could bind to LIN7A, and increased DLG2-isoform 7 gene expression increased the expression of LIN7A, this reduced neuroblastoma cell proliferation and viability, with increased BAX/BCL2 ratio indicating increased apoptosis.ConclusionWe have provided evidence that gene expression of the L27 domain containing DLG2-isoform 7/8 but not L27 domain lacking DLG2-isoform 2 is disrupted in neuroblastoma, in particular in the aggressive subsets of tumors. The presence of the complete L27 domain allows for the binding to LIN7A, which will control cell polarity and signaling, thus affecting cancer cell viability.

Inhibition of the mRNA-Binding Protein IGF2BP1 Suppresses Proliferation and Sensitizes Neuroblastoma Cells to Chemotherapeutic Agents.

Gain at chromosome 17q21 in neuroblastoma is associated with a poor prognosis, independent of MYCN amplification status. Several potential proto-oncogenes have been identified in this region, one of them—insulin-like growth-factor-2 mRNA binding protein (IGF2BP1)—is expressed at high levels in stage 4 tumors, and associated with overall lower patient survival. Here, we demonstrate that down-regulation of IGF2BP1 activity, either by transcript silencing or chemical inhibition, suppresses neuroblastoma cell growth. Furthermore, the combination of IGF2BP1 inhibition along with commonly used chemotherapeutics that broadly affect DNA synthesis, or cyclin-dependent kinase (CDK) inhibitors that disrupt signal transduction, have a synergistic effect on the suppression of neuroblastoma cell proliferation.

EZH2 inhibition decreases neuroblastoma proliferation and in vivo tumor growth.

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted.

Silencing of circular RNA circPDE5A suppresses neuroblastoma progression by targeting the miR-362-5p/NOL4L axis

Objective Neuroblastoma (NB) is the most common extra-cranial solid tumour in early childhood. Circular RNAs (circRNAs) have been implicated in the development of NB. The purpose of the current study was to explore the molecular action of circRNA phosphodiesterase 5 A (circPDE5A) in NB malignant progression. Materials and methods The expression levels of circPDE5A, miR-362-5p and nucleolar protein 4 like (NOL4L) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) assay. Cell migration and invasion were evaluated by transwell assay. The levels of glucose consumption and lactate production were measured using the commercial assay kits. Targeted correlations among circPDE5A, miR-362-5p and NOL4L were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assays were performed to examine the role of circPDE5A in tumour growth in vivo. Results Our results revealed that circPDE5A was up-regulated in NB tissues and cells. The silencing of circPDE5A suppressed NB cell proliferation, migration, invasion, and glycolysis in vitro and diminished tumour growth in vivo. Moreover, circPDE5A directly targeted miR-362-5p by binding to miR-362-5p. CircPDE5A silencing impeded NB malignant progression in vitro through up-regulating miR-362-5p. Furthermore, NOL4L was a direct target of miR-362-5p, and NOL4L mediated the regulation of miR-362-5p on NB malignant progression in vitro. Additionally, circPDE5A functioned as a regulator of NOL4L expression via targeting miR-362-5p. Conclusions Our current findings identified that the knockdown of circPDE5A suppressed NB malignant progression at least in part by the regulation of the miR-362-5p/NOL4L axis, providing a novel rationale for developing circPDE5A as a potential target for NB management.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

The RNA-helicase DDX21 upregulates CEP55 expression and promotes neuroblastoma.

Approximately 25% of human neuroblastoma is caused by amplification of the MYCN oncogene, which leads to overexpression of N‐Myc oncoprotein. The survival rate for this patient subtype is <50%. Here, we show that N‐Myc protein bound to the DEAD‐box RNA helicase DDX21 gene promoter and upregulated DDX21 mRNA and protein expression. Genome‐wide differential gene expression studies identified centrosomal protein CEP55 as one of the genes most dramatically downregulated after DDX21 knockdown in MYCN‐amplified neuroblastoma cells. Knocking down DDX21 or CEP55 reduced neuroblastoma cell cytoskeleton stability and cell proliferation and all but abolished clonogenic capacity. Importantly, DDX21 knockdown initially induced tumor regression in neuroblastoma‐bearing mice and suppressed tumor progression. In human neuroblastoma tissues, a high level of DDX21 expression correlated with a high level of N‐Myc expression and with CEP55 expression, and independently predicted poor patient prognosis. Taken together, our data show that DDX21 induces CEP55 expression, MYCN‐amplified neuroblastoma cell proliferation, and tumorigenesis, and that DDX21 and CEP55 are valid therapeutic targets for the treatment of MYCN‐amplified neuroblastoma.

CircKIF2A contributes to cell proliferation, migration, invasion and glycolysis in human neuroblastoma by regulating miR-129-5p/PLK4 axis.

Multiple circular RNAs (circRNAs) have been identified to act as essential mediators in diverse human cancers. However, the roles of circRNAs in neuroblastoma (NB) are largely unknown. In this study, we aimed to explore the function of circKIF2A in NB. Quantitative real-time polymerase chain reaction was executed to detect the levels of circKIF2A, KIF2A mRNA, miR-129-5p and polo-like kinase 4 (PLK4) mRNA. Actinomycin D assay and RNase R digestion assay were conducted to analyze the feature of circKIF2A. 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay and specific kits were utilized to evaluate cell proliferation, metastasis and glycolysis, respectively. Western blot assay was performed to examine the protein levels of matrix metalloproteinase 2 (MMP2), MMP9 and PLK4. Bioinformatics analysis, RNA pull-down assay and dual-luciferase reporter assay were conducted to analyze the relationship between miR-129-5p and circKIF2A or PLK4. Murine xenograft model assay was done to investigate the role of circKIF2A in NB in vivo. CircKIF2A level was increased in NB tissue samples and cell lines. Silencing of circKIF2A impeded NB cell proliferation, migration, invasion and glycolysis. For mechanism analysis, circKIF2A could positively modulate PLK4 expression via sponging miR-129-5p. Moreover, miR-129-5p inhibition reversed the inhibitory effects of circKIF2A silencing on the behaviors of NB cells. MiR-129-5p overexpression weakened the malignant biological behaviors of NB cells by targeting PLK4. Additionally, circKIF2A knockdown hampered tumorigenesis in vivo. CircKIF2A knockdown suppressed cell proliferation, migration, invasion and glycolysis via downregulating PLK4 expression through miR-129-5p.

Role of estrogen receptor alpha in MEHP-induced proliferation and invasion of SH-SY5Y cells

Estrogen receptors are involved in regulating the proliferation and invasion process of neuroblastoma. As a kind of estrogen-like environmental endocrine disruptors (EEDs), whether mono-2-ethylhexyl phthalate (MEHP) can affect the proliferation and invasion of neuroblastoma cells via ERs is unknown. The present study aimed to explore the role of ERα in MEHP-induced proliferation, migration, and invasion of SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM with 10 % FBS. Wild-type SH-SY5Y cells and ERα-knockdown SH-SY5Y cells were treated with MEHP (0, 10, 50, and 250 μM) for 12 h and 24 h. The viability of SH-SY5Y cells was detected with a CCK8 kit and cell cycle was measured by flow cytometry. Cell migration was measured using a scratch assay, and cell invasion was tested using a Transwell migration assay. The expression levels of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), ERα, and ERβ were detected with real-time qPCR and western blotting. MEHP promoted the proliferation of SH-SY5Y cells. The results also showed that MEHP significantly increased the relative migration distance of wild-type SH-SY5Y cells. Conversely, MEHP treatment did not increase the relative migration distance of ERα-knockdown SH-SY5Y cells, suggesting that MEHP promotes the migration of neuroblastoma through ERα. Similarly, MEHP significantly increased the relative number of invaded wild-type SH-SY5Y cells, while the MEHP-induced invasion effect was significantly decreased in ERα-knockdown SH-SY5Y cells. Moreover, the expression levels of PCNA, MMP-2, MMP-9, and ERα cells were upregulated by MEHP in wild-type SH-SY5Y, and the expression level of its tissue inhibitor TIMP-2 was downregulated. In contrast, the expression of PCNA, MMP-2, MMP-9, and ERα was significantly downregulated in ERα-knockdown SH-SY5Y cells, while the expression of TIMP-2 was significantly upregulated. In conclusion, MEHP can upregulate PCNA, MMP-2, and MMP-9, and downregulate TIMP-2, further promoting proliferation, migration, and invasion of neuroblastoma through ERα.

PTX3 promotes proliferation, invasion and drug resistance of neuroblastoma cells in children by regulating TLR4/NF-κB signaling pathway

Objective: To investigate the effect of pentraxin 3 (PTX3) on the proliferation, invasion and drug resistance of pediatric neuroblastoma cells and its mechanism. Methods: si-RNA (si-RNA group), si-PTX3 (si-PTX3 group), siRNA+ pcDNA3.1 (siRNA+ pcDNA3.1 group), si-PTX3+ pcDNA3.1 (si-PTX3+ pcDNA3.1 group), siRNA+ pcDNA3.1-Toll-like receptor 4 (siRNA+ pcDNA3.1-TLR4 group) and si-PTX3+ pcDNA3.1-TLR4 (si-PTX3+ pcDNA3.1-TLR4 group) were transfected into SH-SY5Y cells. Collected 32 cases of tumor tissue and cancerous tissue in children with childhood neuromaternal cells who were treated at Zhumadian center hospital from July 2016 to August 2019. Real-time fluorescent quantitative polymerase chain (RT-qPCR) reaction and immunohistochemistry experiments were used to detect the protein expressions of PTX3 in neuroblastoma tissues and normal tissues. 5-Ethynyl-2'-deoxyuridine (EdU) was used to detect the proliferation effect of PTX3 on neuroblastoma cell SH-SY5Y. Western blot experiment was used to detect the protein expression levels of vascular endothelial growth factor (VEGF), resistance-related proteins including P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP-1), and invasion-related protein matrix metalloproteinase-1 (MMP-1). Results: PTX3 mRNA expressions in neuroblastoma tissues were 0.87±0.07, higher than 0.13±0.06 of normal tissues, and the differences were statistically significant (P<0.05), The expression of the immunohistochemistry test PTX3 protein was consistent with the qRT-PCR results. Compared with the si-RNA group (0.95±0.08; 1.02±0.10), the mRNA and protein expressions of PTX3 in the si-PTX3 group (0.25±0.05; 0.45±0.66) decreased, the differences were statistically significant (all P<0.05). The number of EdU positive cells, invasion rate, VEGF, MMP-1, P-gp and MRP-1 protein expressions in si-RNA group were (31.86±1.86)%, (28.12±2.96)%, (0.58±0.07), (0.44±0.06), (0.46±0.08) and (0.51±0.05), respectively, higher than (19.73±1.22)%, (8.45±1.06)%, (0.25±0.05), (0.19±0.03), (0.19±0.06) and (0.16±0.07) in si-PTX3 group, and the differences were statistically significant (all P<0.05). The Number of EdU positive cells [(19.49±1.68)%], invasion rate [(8.48±1.36)%], VEGF protein expression (0.10±0.15), P-gp (0.18±0.07) , TLR4 (0.45±0.06), p-p65 (0.25±0.05) protein expressions in si-PTX3+ pcDNA3.1 group were relatively lower compared with siRNA+ pcDNA3.1 group [(38.21±2.67)%, (26.39±2.14)%, 0.49±0.05, 0.52±0.06, 0.93±0.14 and 0.82±0.06] (all P<0.05). The number of EdU-positive cells [(62.73±5.18)%], invasion rate [(50.45±3.25)%], VEGF protein expression (2.17±0.17), P-gp (2.15±0.16), TLR4 (2.68±0.16), p-p65 (2.48±0.13) protein expressions in the siRNA+ pcDNA3.1-TLR4 group increased compared with siRNA+ pcDNA3.1 group (all P<0.05). Conclusions: Inhibition of PTX3 can inhibit the proliferation and invasion of neuroblastoma cells SH-SY5Y, and reduce drug resistance. Its mechanism may be achieved by regulating the TLR4/NF-κB signaling pathway. This result can provide a new perspective for pediatric neuroblasts tumor diagnosis and clinical treatment.

Expression of lncRNA CCAT2 in children with neuroblastoma and its effect on cancer cell growth.

The aim of this study was to determine the expression of long-chain non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) in children with neuroblastoma and its effect on cancer cell growth. A polymerase chain reaction assay was carried out to quantify lncRNA CCAT2 miRNA in neuroblastoma cells, corresponding paracancerous cells, SH-SY5Y and SK-N-SH cells, and human umbilical vein endothelial cells (HUVEC), and two groups of children with different lncRNA CCAT2 expression were compared in clinical pathological parameters and prognosis. CCAT2-NC and si-CCAT2 were transfected into SH-SY5Y cells, separately. Then a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was carried out to analyze the cell proliferation, migration, and invasion ability, a flow cytometry to detect cell apoptosis, and a Western blotting (WB) assay to quantify p53 and Bcl-2 proteins. lncRNA CCAT2 expression in cancer tissues of children with neuroblastoma was notably higher than that in corresponding paracancerous tissues (P < 0.05), and children with different tissue differentiation, tumor staging, and lymph node metastasis (LNM) showed notably different lncRNA CCAT2 expression (P < 0.05). In addition, children with neuroblastoma in the high lncRNA CCAT2 expression group showed lower 3-year survival rate than those in the low expression group (P < 0.05). Multivariate analysis revealed that tissue differentiation, tumor-node-metastasis staging, LNM, and lncRNA CCAT2 expression were all independent risk factors affecting the prognosis of children with neuroblastoma (all P < 0.05). Compared with HUVEC cells, SH-SY5Y and SK-N-SH cells showed notably up-regulated lncRNA CCAT2, and the expression of it in SH-SY5Y was higher than that in SK-N-SH cells (P < 0.05). Compared with the CCAT2-NC group, the si-CCAT2 group presented notably down-regulated CCAT2 (P < 0.05). Moreover, according to the MTT assay, the si-CCAT2 group showed notably weakened cell viability and proliferation than the CCAT2-NC group (both P< 0.05), and SH-SY5Y cells in the former group were less active than those in the latter group in terms of migration and invasion. The cell apoptosis rate of SH-SY5Y cells in the si-CCAT2 was higher than that in the CCAT2-NC. The results suggested that knock down of lncRNA CCAT2 could improve the apoptosis activity of neuroblastoma cells in children. According to the WB assay, the si-CCAT2 group showed notably higher p53 expression and notably lower Bcl-2 protein expression than the CCAT2-NC group (both P < 0.05). LncRNA CCAT2 can inhibit the proliferation of neuroblastoma cells and promote their apoptosis, which provides a basis for the treatment of neuroblastoma.

Poly (C)-Binding Protein 1: A Novel Function Attributed to the Nervous System Microenvironment

Poly (C)-binding protein 1 (PCBP1), which acts as an RNA binding protein, has multiple functions and regulates gene expression by binding to polycytosine, poly (C). The aim of the present study was to investigate the novel function of PCBP1 in the nervous system microenvironmen. The overexpression of PCBP1 has a critical effect on the proliferation and anti-apoptosis of neuroblastoma and glial cells in the direct model, whereas the overexpression of PCBP1 in neuroblastoma or glial cells cannot affect the proliferation or apoptosis of neuroblastoma or glial cells through substances secreted extracellularly. Fur-thermore, through direct or indirect actions, PCBP1 suppressed the expression of nuclear factor kappa B (NF-κB) and the inflammatory cytokine interleukin (IL)-6 and increased the levels of the anti-inflammatory cytokine IL-10; addi-tionally, PCBP1 changed the expression of functional proteins, such as heat shock protein 70 (HSP70), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). The results suggest that PCBP1 can regulate the nervous system in proliferation, apoptosis, inflammatory re-sponse and expression of relevant functional proteins, and it could provide novel targets for gene treating human neurological disorders.

Exportin-T: A Novel Prognostic Predictor and Potential Therapeutic Target for Neuroblastoma.

Exportins as the key mediators of nucleocytoplasmic transport have been identified as the controllers of the passage of numerous types of crucial cancer-related proteins. Targeting exportins in cancer cells might represent an emerging strategy in cancer intervention with the potential to affect clinical outcomes. Here, we focused on the prognostic and therapeutic values of Exportin-T (XPOT) in neuroblastoma. The correlation between the expression and prognostic values of XPOT in patients with neuroblastoma was investigated based on both published transcriptome data and our clinical data. Then, decision curve analysis (DCA) was implemented to identify a XPOT risk prediction model. In addition, RNA inference was performed to silence the expression of XPOT to further investigate the specific roles of XPOT in the progression of neuroblastoma in vitro. Overexpression of XPOT mRNA was associated with poor clinical characteristics, such as age at diagnosis more than 18 months, amplification of MYCN, and advanced International Neuroblastoma Staging System (INSS) stage, and XPOT expression was identified as an independent poor prognosis factor for neuroblastoma using Cox proportional hazards model (P < .001). DCA suggested that neuroblastoma patients could benefit from XPOT risk prediction model-guided interventions (status of MYCN + INSS stage + XPOT). Experimentally, knockdown of XPOT by small interfering RNA inhibited the proliferation and migration in neuroblastoma cells. XPOT is identified as a novel prognostic predictor and potential therapeutic target for neuroblastoma patients. Further investigation should focus on the profound molecular mechanism underlying the tumor inhibition activity of XPOT inhibitors.

The Inhibitory Effects of Olivetol on Cell Proliferation and Invasion of SHSY-5Y Neuroblastoma Cells

The Inhibitory Effects of Olivetol on Cell Proliferation and Invasion of SHSY-5Y Neuroblastoma Cells

Hsa_circ_0013401 Accelerates the Growth and Metastasis and Prevents Apoptosis and Autophagy of Neuroblastoma Cells by Sponging miR-195 to Release PAK2.

Background Increased levels of circRNAs have been identified in a variety of cancers. However, the specific functions and mechanisms of circRNAs in neuroblastoma (NB) have not been fully explored. Methods The levels of hsa_circ_0045997, hsa_circ_0080307, hsa_circ_0013401, hsa_circ_0077578, and microRNA-195 were confirmed by RT-qPCR in NB. Gain- and loss-of-function assays and rescue experiments were conducted to determine the influence of hsa_circ_0013401, miR-195, and P21-activated kinase 2 (PAK2) on the proliferation, apoptosis, autophagy, migration, and invasion of NB cells. Regulatory gene targets were validated by the luciferase assay. A xenograft mouse model was used to determine the in vivo effects of hsa_circ_0013401. Results hsa_circ_0013401 was highly expressed, miR-195 was lowly expressed, and there was a negative correlation between hsa_circ_0013401 and miR-195 in NB. The inhibitory effects of hsa_circ_0013401 knockdown suppressed the proliferation, migration, and invasion and induced the apoptosis and autophagy of NB cells by targeting miR-195 to downregulate PAK2 expression. Luciferase reporter assays showed that miR-195 was a direct target of hsa_circ_0013401, and PAK2 was the downstream target gene of miR-195. In vivo studies showed that hsa_circ_0013401 promotes tumor formation. Conclusions hsa_circ_0013401 induced NB progression through miR-195 to enhance PAK2. Therefore, we might highlight a novel regulatory axis (hsa_circ_0013401/miR-195/PAK2) in NB.