Incidence of Ventriculomegaly in Patients With Craniosynostosis.

Hydrocephalus is variously associated with syndromic craniosynostosis (CS), while it is randomly encountered in nonsyndromic CS. But actually, the ventriculomegaly in CS is less described. In this study, the authors aim to establish whether ventriculomegaly is common in patients with CS, in both syndromic and nonsyndromic. Retrospective measurements of Evans index (EI) were taken from thin-section computed tomography scans of 169 preoperative CS patients to assess cerebral ventricular volume. EI >0.3 indicates ventricular enlargement. A total of 169 CS patients who underwent computed tomography scan from February 2018 to December 2021 were retrospectively evaluated, including 114 males and 55 females. The average age at diagnosis was 16 months (range: 1-103 mo). Among them, 37 with syndromic CS, including 17 ventricular megaly patients, had an EI >0.3 (46.0%), and 4 of them had intracranial hypertension and needed ventriculoperitoneal shunt treatment before cranial vault remolding. One hundred and thirty-two had nonsyndromic CS (100 single-suture CS, 32 multisuture CS), and 26 of them had an EI of 0.3 or greater (19.7%). Ventrocular megaly is common among patients with CS. Early craniotomy may stabilize ventricular dilation.

MYBL2 accelerates epithelial-mesenchymal transition and hepatoblastoma metastasis via the Smad/SNAI1 pathway.

Hepatoblastoma (HB) accounts for the majority of hepatic malignancies in children. Although the prognosis of patients with HB has improved in past decades, metastasis is an indicator of poor overall survival. Herein, we applied single-cell RNA sequencing to explore the transcriptomic profiling of 25,264 metastatic cells isolated from the lungs of two patients with HB. The transcriptomes uncovered the heterogeneity of malignant cells after metastatic lung colonization, and these cells had varied expression signatures associated with the cell cycle, epithelial-mesenchymal plasticity, and hepatic differentiation. Single-cell regulatory network inference and clustering (SCENIC) was utilized to identify the co-expressed transcriptional factors which regulated and represented the different cell states. We further screened the key factor by bioinformatics analysis and found that MYBL2 upregulation was significantly associated with metastasis and poor prognosis. The relationship between ectopic MYBL2 and metastasis was subsequently proved by immunohistochemistry (IHC) of HB tissues, and the functions of MYBL2 in promoting proliferation, migration, and epithelial-to-mesenchymal transition (EMT) were verified by in vitro and in vivo assays. Importantly, the levels of Smad2/3 phosphorylation and SNAI1 expression were increased in MYBL2-transfected cells. Consequently, these results indicated that the MYBL2-controlled Smad/SNAI1 pathway induced EMT and promoted HB tumorigenesis and metastasis.

Tumor Suppressive Role of MUC6 in Wilms Tumor via Autophagy-Dependent ß-Catenin Degradation.

Wilms tumor is the most common renal malignancy in children. Known gene mutations account for about 40% of all wilms tumor cases, but the full map of genetic mutations in wilms tumor is far from clear. Whole genome sequencing and RNA sequencing were performed in 5 pairs of wilms tumor tissues and adjacent normal tissues to figure out important genetic mutations. Gene knock-down, CRISPR-induced mutations were used to investigate their potential effects in cell lines and in-vivo xenografted model. Mutations in seven novel genes (MUC6, GOLGA6L2, GPRIN2, MDN1, MUC4, OR4L1 and PDE4DIP) occurred in more than one patient. The most prevalent mutation was found in MUC6, which had 7 somatic exonic variants in 4 patients. In addition, TaqMan assay and immunoblot confirmed that MUC6 expression was reduced in WT tissues when compared with control tissues. Moreover, the results of MUC6 knock-down assay and CRISPR-induced MUC6 mutations showed that MUC6 inhibited tumor aggression via autophagy-dependent ß-catenin degradation while its mutations attenuated tumor-suppressive effects of MUC6. Seven novel mutated genes (MUC6, GOLGA6L2, GPRIN2, MDN1, MUC4, OR4L1 and PDE4DIP) were found in WT, among which MUC6 was the most prevalent one. MUC6 acted as a tumor suppressive gene through autophagy dependent ß-catenin pathway.

m6A Modification: A Double-Edged Sword in Tumor Development.

Modification of m6A, as the most abundant mRNA modification, plays diverse roles in various biological processes in eukaryotes. Emerging evidence has revealed that m6A modification is closely associated with the activation and inhibition of tumor pathways, and it is significantly linked to the prognosis of cancer patients. Aberrant reduction or elevated expression of m6A regulators and of m6A itself have been identified in numerous tumors. In this review, we give a description of the dynamic properties of m6A modification regulators, such as methyltransferases, demethylases, and m6A binding proteins, and indicate the value of the balance between these proteins in regulating the expression of diverse genes and the underlying effects on cancer development. Furthermore, we summarize the "dual-edged weapon" role of RNA methylation in tumor progression and discuss that RNA methylation can not only result in tumorigenesis but also lead to suppression of tumor formation. In addition, we summarize the latest research progress on small-molecule targeting of m6A regulators to inhibit or activate m6A. These studies indicate that restoring the balance of m6A modification via targeting specific imbalanced regulators may be a novel anti-cancer strategy.

[Corrigendum] Linc01105 acts as an oncogene in the development of neuroblastoma.

Subsequently to the publication of the above article, the authors have found that Fig. 4A on p. 1532 contained some errors. Owing to mistakes made during the preparation and revision of the manuscript, the invasion assay data images selected to show both the 'Control' and 'shRNA2' groups of the invasion and migration experiments were derived from the same original sources. A corrected version of the Fig. 4, showing the correct data for the invasion and migration assay experiments with the Control and shRNA2 groups, is shown below. These inadvertent errors did not affect the conclusions reported in this paper, and all the authors agree with this Corrigendum. The authors thank the editor of Oncology Reports for presenting them with the opportunity to publish this Corrigendum, and apologize to the editor and to the readership of the journal for any inconvenience caused. [the original article was published in Oncology Reports 42: 1527-1538, 2019; DOI: 10.3892/or.2019.7257].

USP7 promotes hepatoblastoma progression through activation of PI3K/AKT signaling pathway.

BACKGROUND: Hepatoblastoma (HB) is an embryonic solid tumor and the most common primary malignant liver tumor in children. HB usually occurs in infants and children. Although treatment diversity is increasing, some patients still have very poor prognosis. Many studies have investigated USP7 inhibitors for tumors. Using database information, we found that USP7 is highly expressed in HB. METHODS: Lentivirus-mediated USP7 knockdown and overexpression was performed in HB cell lines HepG2 and Huh6. CCK8 and transwell assays were used to determine cell viability and metastasis. Flow cytometry was used to study cell cycle and apoptosis. Levels of proteins were detected using western blots. RESULTS: Downregulation of USP7 resulted in significant decrease in cell proliferation, clonal formation, and cell migration and invasion. With overexpression of USP7, cellular malignant behavior increased. Cell cycle assays showed that USP7 knockdown inhibited G1 to S phase transition in the cell cycle. Upregulation of USP7 promoted the transition. Animal experiments showed USP7 facilitated tumor growth in vivo. Western blots indicated that USP7 may affect HB tumorigenesis through the PI3K/AKT signaling pathway. Furthermore, USP7 inhibitor P5091 inhibited HB development and PI3K/AKT pathway. CONCLUSION: USP7 upregulation contributed to HB genesis and development through the PI3K/AKT signaling pathway. USP7 could be a potential target for future HB treatment.

ARID1B/SUB1-activated lncRNA HOXA-AS2 drives the malignant behaviour of hepatoblastoma through regulation of HOXA3.

It has been becoming increasingly evident that long non-coding RNAs (lncRNAs) play important roles in various human cancers. However, the biological processes and clinical significance of most lncRNAs in hepatoblastoma (HB) remain unclear. In our previous study, genome-wide analysis with a lncRNA microarray found that lncRNA HOXA-AS2 was up-regulated in HB. Stable transfected cell lines with HOXA-AS2 knockdown or overexpression were constructed in HepG2 and Huh6 cells, respectively. Our data revealed knockdown of HOXA-AS2 increased cell apoptosis and inhibited cell proliferation, migration and invasion in HB. Up-regulation of HOXA-AS2 promoted HB malignant biological behaviours. Mechanistic investigations indicated that HOXA-AS2 was modulated by chromatin remodelling factor ARID1B and transcription co-activator SUB1, thereby protecting HOXA3 from degradation. Therefore, HOXA-AS2 positively regulates HOXA3, which might partly demonstrate the involvement of HOXA3 in HOXA-AS2-mediated HB carcinogenesis. In conclusion, HOXA-AS2 is significantly overexpressed in HB and the ARID1B/HOXA-AS2/HOXA3 axis plays a critical role in HB tumorigenesis and development. These results might provide a potential new target for HB diagnosis and therapy.

Role of long noncoding RNA in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite a variety of treatments, patients with advanced stage disease still have a poor prognosis. The molecular mechanisms underlying NB pathogenesis are not fully known. Increasing evidence shows that long noncoding RNA (lncRNA) is important in multiple ways in NB progression. LncRNA could act as competitive endogenous RNA, serve as a scaffold for proteins, or participate in histone modification, thus affecting proliferation, migration, invasion, and differentiation of NB. Numerous lncRNAs polymorphisms are significantly associated with NB susceptibility. Differently expressed lncRNAs can be used to construct risk scores to evaluate patient outcomes. In conclusion, these lncRNAs have the potential to be diagnostic markers as well as promising therapeutic targets in the future.

S119N Mutation of the E3 Ubiquitin Ligase SPOP Suppresses SLC7A1 Degradation to Regulate Hepatoblastoma Progression.

A previous study on hepatoblastoma revealed novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex, including the tumor suppressor speckle-type BTB/POZ (SPOP). Moreover, the SPOP gene affected cell growth, and its S119N mutation was identified as a loss-of-function mutation in hepatoblastoma. This study aimed to explore more functions and the potential mechanism of SPOP and its S119N mutation. The in vitro effects of SPOP on cell proliferation, invasion, apoptosis, and in vivo tumor growth were investigated by western blot analysis, Cell Counting Kit-8, colony formation assay, flow cytometry, and xenograft animal experiments. The substrate of SPOP was discovered by a protein quantification assay and quantitative ubiquitination modification assay. The present study further proved that SPOP functioned as an anti-oncogene through the phosphatidylinositol 3-kinase/Akt signaling pathway to affect various malignant biological behaviors of hepatoblastoma both in vitro and in vivo. Furthermore, experimental results also suggested that solute carrier family 7 member 1 (SLC7A1) might be a substrate of SPOP and influence cell phenotype by regulating arginine metabolism. In conclusion, these findings demonstrated the function of SPOP and revealed a potential substrate related to hepatoblastoma tumorigenesis, which might thus provide a novel therapeutic target for hepatoblastoma.

Emerging role of long noncoding RNA-encoded micropeptides in cancer.

Increasing evidence has indicated that long noncoding RNAs (lncRNAs) play various important roles in the development of cancers. The widespread applications of ribosome profiling and ribosome nascent chain complex sequencing revealed that some short open reading frames of lncRNAs have micropeptide-coding potential. The resulting micropeptides have been shown to participate in N6-methyladenosine modification, tumor angiogenesis, cancer metabolism, and signal transduction. This review summarizes current information regarding the reported roles of lncRNA-encoded micropeptides in cancer, and explores the potential clinical value of these micropeptides in the development of anti-cancer drugs and prognostic tumor biomarkers.

Competing endogenous RNA network analysis for screening inflammation­related long non­coding RNAs for acute ischemic stroke.

Long non­coding RNAs (lncRNAs) represent potential biomarkers for the diagnosis and treatment of various diseases; however, the role of circulating acute ischemic stroke (AIS)­related lncRNAs remains relatively unknown. The present study aimed to screen crucial lncRNAs for AIS based on the competing endogenous RNA (ceRNA) hypothesis. The expression profile datasets for one mRNA, accession no. GSE16561, and four microRNAs (miRNAs), accession nos. GSE95204, GSE86291, GSE55937 and GSE110993, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified, and ClusterProfiler was used to interpret the function of the DEGs. Based on the protein­protein interaction (PPI) network and module analyses, hub DEGs were identified. A ceRNA network was established based on miRNA­mRNA or miRNA­lncRNA interaction pairs. In total, 2,041 DEGs and 5 DELs were identified between the AIS and controls samples in GSE16561, and 10 DEMs between at least two of the four miRNA expression profiles. A PPI network was constructed with 1,235 DEGs, among which 20 genes were suggested to be hub genes. The hub genes paxillin (PXN), FYN­proto­oncogene, Src family tyrosine kinase (FYN), ras homolog family member A (RHOA), STAT1, and growth factor receptor­bound protein 2 (GRB2), were amongst the most significantly enriched modules extracted from the PPI network. Functional analysis revealed that these hub genes were associated with inflammation­related signaling pathways. An AIS­related ceRNA network was constructed, in which 4 DELs were predicted to function as ceRNAs for 9 DEMs, to regulate the five identified hub genes; that is, minichromosome maintenance complex component 3 associated protein­antisense RNA 1 (MCM3AP­AS1)/long intergenic non­protein coding RNA 1089 (LINC01089)/hsa­miRNA (miR)­125a/FYN, inositol­tetrakisphosphate 1­kinase­antisense RNA 1 (ITPK1­AS1)/hsa­let­7i/RHOA/GRB2/STAT1, and human leukocyte antigen complex group 27 (HCG27)/hsa­-miR­19a/PXN interaction axes. In conclusion, MCM3AP­AS1, LINC01089, ITPK1­AS1, and HCG27 may represent new biomarkers and underlying targets for the treatment of AIS.

Single-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma.

Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB.

Determination of long non-coding RNAs associated with EZH2 in neuroblastoma by RIP-seq, RNA-seq and ChIP-seq.

Neuroblastoma (NB) is the most common type of extracranial solid tumor found in children. Despite several treatment options, patients with advanced stage disease have a poor prognosis. Previous studies have reported that enhancer of zeste homolog 2 (EZH2) and long non-coding RNAs (lncRNAs) have abnormal expression levels in NB and participate in tumorigenesis and NB development. However, the association between EZH2 and lncRNAs remain unclear. In the present study, RNA immunoprecipitation-sequencing (RIP-seq) was used to analyze the lncRNAs binding to EZH2. Following EZH2 knockdown via short hairpin RNA, RNA-seq was performed in shEZH2 and control groups in SH-SY5Y cells. Chromatin IP (ChIP)-seq was used to determine the genes that may be regulated by EZH2. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the signaling pathways involved in NB. The results from RIP-seq identified 94 lncRNAs, including SNHG7, SNHG22, KTN-AS1 and Linc00843. Furthermore, results from RNA-seq demonstrated that, following EZH2 knockdown, 448 genes were up- and 571 genes were downregulated, with 32 lncRNAs up- and 35 downregulated and differentially expressed compared with control groups. Certain lncRNAs, including MALAT1, H19, Linc01021 and SNHG5, were differentially expressed in EZH2-knockdown group compared with the control group. ChIP-seq identified EZH2 located in the promoter region of 138 lncRNAs including CASC16, CASC15, LINC00694 and TBX5-AS1. In summary, the present study demonstrated that certain lncRNAs directly bound EZH2 and regulated EZH2 expression levels. A number of these lncRNAs that are associated with EZH2 may participate in NB tumorigenesis.

Linc01105 acts as an oncogene in the development of neuroblastoma.

Previous research from our group revealed that the long coding RNA (lncRNA) linc01105 is associated with neuroblastoma proliferation and apoptosis, and that its expression is correlated with the International Neuroblastoma Staging System stage. The purpose of the present study was to investigate the functions of Linc01105 in neuroblastoma. Lentivirus­mediated linc01105 knockdown was performed in the neuroblastoma cell line SH­SY5Y. The expression levels of linc01105 and of other associated genes were measured by reverse transcription­quantitative PCR. Cell Counting Kit­8 assay and flow cytometry were used to determine cell viability and apoptosis. The levels of proteins were detected using western blot analysis. Bioinformatics analysis and luciferase reporter assays were used to examine the relationship between linc01105, miR­6769b­5p and vascular endothelial growth factor A (VEGFA). Angiogenesis ability was measured using a tube formation assay. The results demonstrated that HIF­1α overexpression promoted the transcription of linc01105 by acting as a transcription factor. Knockdown of linc01105 inhibited neuroblastoma cell proliferation, migration and invasion, and it induced apoptosis. In addition, linc01105 affected the expression of p53 and Bcl­2 family proteins and activated the caspase signaling pathway. Further functional experiments revealed that linc01105 promoted the expression of the miR­6769b­5p target gene VEGFA by acting as a sponge of miR­6769b­5p. In conclusion, linc01105 may contribute to neuroblastoma tumorigenesis and development. The present findings indicated that the interplay between the p53/caspase pathway and the linc01105/miR­6769b­5p/VEGFA axis may have important roles in the development of neuroblastoma.

Mesoporous silica nanoparticles as a delivery system for improving antiangiogenic therapy.

PURPOSE: Antiangiogenic drugs usually have short-acting efficacy and poor treatment compliance. The purpose of this study was to determine whether mesoporous silica nanoparticles (MSNs) could be utilized as a nanodrug delivery system for improving antiangiogenic therapy. MATERIALS AND METHODS: MSN-encapsulated bevacizumab nanoparticles were prepared by the nanocasting strategy and characterized by Fourier transform infrared, transmission electron microscopy, and Brunauer-Emmett-Teller method. Encapsulation efficiency and drug loading efficiency of MSN-encapsulated bevacizumab nanoparticles were calculated. The pharmacokinetics, cytotoxicity, and tissue toxicity were evaluated in vitro and in vivo. The antiangiogenic effects of MSN-bevacizumab nanoparticles were evaluated in vitro and in vivo. RESULTS: MSN encapsulation could prolong the residency of bevacizumab in vitreous/aqueous humor and maintain the long-lasting drug concentration. MSN-encapsulated bevacizumab nanoparticles did not show any obvious cytotoxicity and tissue toxicity. MSN-encapsulated bevacizumab nanoparticles were more effective than bevacizumab in suppressing vascular endothelial growth factor-induced endothelial cell proliferation, migration, and tube formation in vitro. MSN-encapsulated bevacizumab nanoparticles showed sustained inhibitory effects on corneal neovascularization and retinal neovascularization in vivo. CONCLUSION: This study provides a novel strategy of encapsulating bevacizumab to protect and deliver it, which could increase the time between administration and formulation shelf-life. MSN-encapsulated bevacizumab is a promising drug delivery alternative of antiangiogenic therapy.

Effect of nanoencapsulation using poly (lactide-co-glycolide) (PLGA) on anti-angiogenic activity of bevacizumab for ocular angiogenesis therapy.

Antibody-based therapy is an effective strategy for treating ocular angiogenesis. However, short-acting efficacy and poor treatment compliance usually occurs in clinical practices. Thus, it is required to develop a drug delivery system to improve the bioavailability and decrease the toxicity of anti-angiogenic antibody. Bevacizumab is a recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGF). In this study, bevacizumab was encapsulated into poly (lactide-co-glycolide) (PLGA) nanoparticles. PLGA encapsulation could prolong the residency of bevacizumab in the vitreous and aqueous humor and produce long-lasting drug concentrations. Bevacizumab-encapsulated PLGA had no significant cytotoxicity and tissue toxicity effect in vitro and in vivo. In vitro studies showed that bevacizumab-encapsulated PLGA was more effective than bevacizumab in inhibiting VEGF-mediated endothelial cell proliferation, migration and tube formation. In vivo studies showed that bevacizumab-encapsulated PLGA enhanced the anti-angiogenic efficiency of bevacizumab for treating corneal neovascularization and retinal neovascularization. Thus, bevacizumab-encapsulated PLGA could increase the bioavailability and decrease the toxicity of bevacizumab during ocular angiogenesis therapy.

Circulating tumor cells detection in neuroblastoma patients by EpCAM-independent enrichment and immunostaining-fluorescence in situ hybridization.

Detecting circulating tumor cells (CTCs) has proven valuable for evaluating the prognosis of cancer patients and for studying the mechanisms of treatment resistance. Owing to the lack of universal and specific tumor markers for neuroblastoma (NB), in this prospective study, we adopted an EpCAM-independent method to detect CTCs in the peripheral blood of NB patients. We used an EpCAM-independent assay to delete leukocytes and to enrich the CTCs. CTCs were identified by immunostaining of CD45, DAPI and DNA fluorescence in situ hybridization (FISH) of the centromere of chromosome 8 probe (CEP8). Cells that were DAPI+/CD45-/CEP8 ≥ 3 were considered CTCs. We collected peripheral blood from 28 NB patients as well as clinical and follow-up data. The number of CTCs among the different risk groups were significantly different (p = .0208, Kruskal-Wallis test). Patients with metastasis had more CTCs than those without metastasis (p < .0001, Mann-Whitney test). Patients with ≥3 CTCs per 4 ml of peripheral blood had an increased likelihood of having metastasis (sensitivity, 88.89%; specificity, 78.59%), and patients with ≥10 CTCs per 4 ml of peripheral blood had poorer overall survival. The EpCAM-independent assay along with immunostaining-FISH (i-FISH) described here can detect CTCs in patients with NB at a high sensitivity and may have clinical value for prognosis evaluation and diagnosing metastasis when imaging data are ambiguous.

Circular RNA HIPK3 regulates human lens epithelial cells proliferation and apoptosis by targeting the miR-193a/CRYAA axis.

Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back splicing. Accumulating evidence has demonstrated their vital regulation in several biological processes and ocular diseases. However, the role of circRNAs in age-related cataract (ARC), the leading cause of visual impairment worldwide, is still unknown. CircRNA sequencing reveals that 101 circRNAs are differentially expressed between the capsules of transparent and ARC lenses, including 75 down-regulated circRNAs and 26 up-regulated circRNAs transcripts. Eight of 10 differentially expressed circRNAs are further verified by quantitative RT-PCRs. One highly conserved circRNA, circHIPK3, is significantly down-regulated in all cortical, nuclear and posterior subcapsular subtypes of ARC. The silencing of circHIPK3, but not HIPK3 mRNA, significantly accelerates apoptosis development upon oxidative stress and decreases cell viability and proliferation in primary cultured human lens epithelial cells (HLECs). The expression of α-SMA and vimentin was downregulated, while the expression of E-cadherin and ZO-1was upregulated, suggesting the repression of epithelial-mesenchymal transition after circHIPK3 knockdown. CircHIPK3 silencing increases miR-193a expression. miR-193a regulates CRYAA expression by targeting the binding site within the 3'UTR. Moreover, miR-193a decreases the viability and proliferation, and increases the apoptosis of HLECs upon oxidative stress. This study suggests that circRNAs are the potential regulators in cataractogenesis. CircHIPK3 regulates HLECs function through miR-193a-mediated CRYAA expression. This finding would provide a novel insight into the pathogenesis of ARC.