Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Netrin1 deficiency activates MST1 via UNC5B receptor, promoting dopaminergic apoptosis in Parkinson's disease.

The Hippo (MST1/2) pathway plays a critical role in restricting tissue growth in adults and modulating cell proliferation, differentiation, and migration in developing organs. Netrin1, a secreted laminin-related protein, is essential for nervous system development. However, the mechanisms underlying MST1 regulation by the extrinsic signals remain unclear. Here, we demonstrate that Netrin1 reduction in Parkinson's disease (PD) activates MST1, which selectively binds and phosphorylates netrin receptor UNC5B on T428 residue, promoting its apoptotic activation and dopaminergic neuronal loss. Netrin1 deprivation stimulates MST1 activation and interaction with UNC5B, diminishing YAP levels and escalating cell deaths. Knockout of UNC5B abolishes netrin depletion-induced dopaminergic loss, whereas blockade of MST1 phosphorylating UNC5B suppresses neuronal apoptosis. Remarkably, Netrin1 is reduced in PD patient brains, associated with MST1 activation and UNC5B T428 phosphorylation, which is accompanied by YAP reduction and apoptotic activation. Hence, Netrin1 regulates Hippo (MST1) pathway in dopaminergic neuronal loss in PD via UNC5B receptor.

Identification and validation of HOXD3 and UNC5C as molecular signatures in keloid based on weighted gene co-expression network analysis.

BACKGROUND: Keloid is a benign proliferative disease characterized by excessive deposition of extracellular matrix collagen during skin wound healing. The mechanisms of keloid formation have not been fully elucidated, and the current treatment methods are not effective for all keloid patients. Therefore, there is an urgent need to find more effective therapies, and our research focused on identifying characteristic molecular signatures of keloid to explore potential therapeutic targets. METHODS: Gene expression profiles of keloid and control group samples were retrieved from the GEO database. Taking the GSE113619 dataset as the training set, the dataset collected skin tissues from non-lesion sites of healthy and keloid-prone individuals, denoted as Day0. The second sampling was performed 42 days later at the original sampling site of control and keloid groups, denoted as Day42.The 'limma' package and Venn diagram identified differentially expressed genes (DEGs) specific to keloid day42 versus day0 samples. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathway functional enrichment, and annotation of the characteristic genes were conducted on the Metascape website. Ingenuity canonical pathways, disease & function enrichment analysis and gene interaction network were performed and predicted in Ingenuity Pathway Analysis (IPA) software. Key module genes related to keloid were filtered out by Weighted Gene Co-expression Network Analysis (WGCNA). We utilized the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm to screen the characteristic genes in keloid by the 'glmnet' package. The area under the curve (AUC) of receiver operating characteristic (ROC) was utilized to determine the effectiveness of potential signatures in discriminating keloid samples from normal samples and performed by using the 'pROC' package. The enrich scores of 24 immune cells in each sample were calculated by the single-sample gene set enrichment analysis (ssGSEA) algorithm， and then the Gene Set Variation Analysis (GSVA) was performed. Finally, RNA from 4 normal and 6 keloid samples was extracted, and RT-qPCR and Western Blot validated the expression of characteristic genes. RESULTS: A total of 640 DEGs specific to keloid day42 versus day0 samples were detected. 69 key module genes were uncovered and implicated in 'NCAM signaling for neurite out-growth', 'oncogenic MAPK signaling', 'transmission across chemical synapses' pathways, and the mitotic cell cycle-related processes. Five characteristic genes (MTUS1, UNC5C, CEP57, NAA35, and HOXD3) of keloid were identified by LASSO, and among which UNC5C and HOXD3 were validated by ROC plot in external dataset, RT-qPCR and Western Blot in validation samples. The result of ssGSEA indicated that the infiltration of neutrophils showed a relatively higher abundance and natural killer cells with relatively low enrichment in the keloid group compared to the control group. UNC5C was correlated with more immune cells compared with other characteristic genes. CONCLUSION: In this study, characteristic genes associated with keloid were identified by bioinformatic approaches and verified in clinical validation samples, providing potential targets for the diagnosis and treatment of keloid.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism.

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2'-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors.

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.

The intracellular domain of UNC5B facilities proliferation and metastasis of bladder cancer cells.

The intracellular domain of UNC5B contains both death domain and caspase-3 cleavage site, and is regarded as a functional domain that mediates apoptosis. However, in our previous studies, we found that the death domain of UNC5B in bladder cancer cells could not be activated to promote apoptosis. In this study, different UNC5B truncates (residue 399-945, residue 412-945) were created to explore whether the caspase-3 cleavage site (site 412), as another potential functional domain of its intracellular portion, could be activated to induce apoptosis in bladder cancer cells. Using mass spectrometry, we acquired a comprehensive and detailed identification of differentially expressed proteins by overexpressing UNC5B and its truncates. Protein-protein-interaction (PPI) network analysis was also applied to investigate the aggregation of related proteins and predict the functional changes. EDU assay, apoptosis, xenograft tumour implantation, migration, invasion and tumour metastasis were performed to comprehensively identify the effects of UNC5B truncates on bladder cancer cells. We demonstrate that the intracellular domain of UNC5B promotes cell proliferation in vitro and tumour formation in vivo, by binding to a large number of ribosomal proteins. The overexpression of intracellular domain also facilitates cells to migrate, invade and metastasize by interacting with fibronectin, beta-catenin and vimentin. In addition, we reveal that overexpressing the intracellular domain of UNC5B cannot bind or activate cleaved caspase-3 to trigger apoptosis in bladder cancer cells.

Inhibition of the long non-coding RNA UNC5B-AS1/miR-4455/RSPO4 axis reduces cervical cancer growth in vitro and in vivo.

BACKGROUND: Long non-coding RNAs (lncRNAs) are significant regulatory factors for the initiation and development of numerous malignant tumors, including cervical cancer (CC). The expression of lncRNA unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1, also known as UASR1) is up-regulated in tissues of cervical squamous cell carcinoma and endocervical adenocarcinoma compared to in normal tissues based on the GEPIA database. In the present study, we explored the functions of UNC5B-AS1 and its underlying mechanism with respect to CC development. METHODS: A real-time quantitative polymerase chain reaction was applied for the detection of UNC5B-AS1 expression in CC cells. Cell counting kit-8, colony formation and transwell assays, as well as western blot and flow cytometry analyses, were employed to detect the biological effects of UNC5B-AS1 knockdown on malignant phenotypes of CC cells in vitro. In addition, the combination between microRNA-4455 (miR-4455) and UNC5B-AS1 or R-spondin 4 (RSPO4) was explored by RNA immunoprecipitation, luciferase reporter and RNA pulldown assays. A tumor xenograft nude mice model was established to explore the effect of UNC5B-AS1 depletion or miR-4455 overexpression on tumor growth. RESULTS: UNC5B-AS1 is up-regulated in CC tissues and cells. The knockdown of UNC5B-AS1 inhibits CC cell proliferation, migration and invasion and promotes CC cell apoptosis. Mechanistically, UNC5B-AS1 binds with miR-4455 to up-regulate RSPO4 expression. RSPO4 is targeted by miR-4455 and its expression is negatively regulated by miR-4455 expression. In vivo assays revealed that UNC5B-AS1 depletion or miR-4455 overexpression inhibits tumor growth by regulating RSPO4 expression. CONCLUSIONS: Inhibition of UNC5B-AS1/miR-4455/RSPO4 reduces CC growth both in vitro and in vivo, furnishing new insights into molecular studies on UNC5B-AS1 with respect to CC development.

Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner.

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A.IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.

Ischemia-induced Netrin-4 promotes neovascularization through endothelial progenitor cell activation via Unc-5 Netrin receptor B.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

Brain Tumor promotes axon growth across the midline through interactions with the microtubule stabilizing protein Apc2.

Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or fra mutants, the loss of crossing is incomplete, suggesting the existence of additional pathways. Here, we identify Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the Drosophila CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, we show that through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, we propose that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.

Netrin-1 in Glioblastoma Neovascularization: The New Partner in Crime?

Glioblastoma (GBM) is the most aggressive and common primary tumor of the central nervous system. It is characterized by having an infiltrating growth and by the presence of an excessive and aberrant vasculature. Some of the mechanisms that promote this neovascularization are angiogenesis and the transdifferentiation of tumor cells into endothelial cells or pericytes. In all these processes, the release of extracellular microvesicles by tumor cells plays an important role. Tumor cell-derived extracellular microvesicles contain pro-angiogenic molecules such as VEGF, which promote the formation of blood vessels and the recruitment of pericytes that reinforce these structures. The present study summarizes and discusses recent data from different investigations suggesting that Netrin-1, a highly versatile protein recently postulated as a non-canonical angiogenic ligand, could participate in the promotion of neovascularization processes in GBM. The relevance of determining the angiogenic signaling pathways associated with the interaction of Netrin-1 with its receptors is posed. Furthermore, we speculate that this molecule could form part of the microvesicles that favor abnormal tumor vasculature. Based on the studies presented, this review proposes Netrin-1 as a novel biomarker for GBM progression and vascularization.

UNC5B-AS1 promoted ovarian cancer progression by regulating the H3K27me on NDRG2 via EZH2.

The role of long non-coding RNAs (lncRNAs) in tumorigenesis and development of ovarian cancer (OC) has caught the attention of scientists. UNC5B antisense RNA 1 (UNC5B-AS1) is a newly identified carcinogenic lncRNA in thyroid papillary carcinoma, but its role in OC remains unclear. This study is proposed to investigate the function and mechanism of UNC5B-AS1 in OC. UNC5B-AS1 expression in OC samples was obtained from gene expression profiling interactive analysis (GEPIA) based on The Cancer Genome Atlas data. Gene expressions were detected by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. Biological functions of UNC5B-AS1 were assessed by cell counting kit-8, colony formation, and caspase-3 analysis. GEPIA revealed the UNC5B-AS1 upregulation in OC samples. RT-qPCR assay confirmed the upregulation of UNC5B-AS1 in OC cells. Functionally, depletion of UCN5B-AS1 hindered proliferation and prompted apoptosis in OC cells. Mechanistically, we found that UNC5B-AS1 interacted with zeste 2 polycomb repressive complex 2 subunit (EZH2) to trigger trimethylation of histone H3 at lysine 27 (H3K27me3) on N-myc downstream regulated gene-2 (NDRG2) promoter and epigenetically repressed NDRG2. Rescue assay indicated the participation of NDRG2 in the regulation of UNC5B-AS1 on OC progression. Together, we first illustrated that UNC5B-AS1 promoted OC progression by regulating the H3K27me on NDRG2 via EZH2, indicating UNC5B-AS1 as a potential molecular target for OC treatment.

Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3.

Thrombospondin type 1 repeats (TSRs) occur in diverse proteins involved in adhesion and signaling. The two extracellular TSRs of the netrin receptor UNC5A contain WxxWxxWxxC motifs that can be C-mannosylated on all tryptophans. A single C-mannosyltransferase (dumpy-19, DPY-19), modifying the first two tryptophans, occurs in Caenorhabditis elegans, but four putative enzymes (DPY-19-like 1-4, DPY19L1-4) exist in mammals. Single and triple CRISPR-Cas9 knockouts of the three homologs that are expressed in Chinese hamster ovary cells (DPY19L1, DPY19L3, and DPY19L4) and complementation experiments with mouse homologs showed that DPY19L1 preferentially mannosylates the first two tryptophans and DPY19L3 prefers the third, whereas DPY19L4 has no function in TSR glycosylation. Mannosylation by DPY19L1 but not DPY19L3 is required for transport of UNC5A from the endoplasmic reticulum to the cell surface. In vertebrates, a new C-mannosyltransferase has apparently evolved to increase glycosylation of TSRs, potentially to increase the stability of the structurally essential tryptophan ladder or to provide additional adhesion functions.

Netrin-1 alleviates subarachnoid haemorrhage-induced brain injury via the PPARγ/NF-KB signalling pathway.

Netrin-1 (NTN-1) is a novel drug to alleviate early brain injury following subarachnoid haemorrhage (SAH). However the molecular mechanism of NTN-1-mediated protection against early brain injury following SAH remains largely elusive. This study aims to evaluate the effects and mechanisms of NTN-1 in protecting SAH-induced early brain injury. The endovascular perforation SAH model was constructed using male C57BL/6J mice, and recombinant NTN-1 was administrated intravenously. Mortality rates, SAH grade, brain water content, neurological score and neuronal apoptosis were evaluated. The expression of PPARγ, Bcl-2, Bax and nuclear factor-kappa B (NF-κB) were detected by Western blot. Small interfering RNA specific to NTN-1 receptor, UNC5B, and a selective PPARγ antagonist, bisphenol A diglycidyl ether (BADGE), were applied in combination with NTN-1. The results suggested that NTN-1 improved the neurological deficits, reduced the brain water content and alleviated neuronal apoptosis. In addition, NTN-1 enhanced PPARγ and Bcl-2 expression and decreased the levels of Bax and NF-κB. However, the neuroprotection of NTN-1 was abolished by UNC5B and BADGE. In conclusion, our results demonstrated that NTN-1 attenuates early brain injury following SAH via the UNC5B PPARγ/NF-κB signalling pathway.

The role of UNC5b in ox-LDL inhibiting migration of RAW264.7 macrophages and the involvement of CCR7.

The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. The C-C chemokine receptor type 7 (CCR7) is positively correlated with the macrophage migration. But the mechanism of CCR7 regulation is not fully clearness. In the present study, we demonstrates that expression in UNC5b and netrin-1 was enhanced in respond to ox-LDL in Raw264.7 macrophage and associated with decreasing cell migration. Interestingly, it was found that ox-LDL significantly downregulate CCR7 gene expression. The expression of CCR7 in mRNA and protein levels were decreased in ox-LDL treated Raw264.7 macrophage when we over expression of UNC5b with pcDNA3.1-UNC5b plasmid. We got the inverse results after silence UNC5b gene with siUNC5b. Meanwhile, the data show that in ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. This novel phenomenon is of a crucial highlights to understand deeply the pathogenesis of atherosclerosis. The molecular mechanism of CCR7 regulation deserves intensive study.

UNC5C: Novel Gene Associated with Psychiatric Disorders Impacts Dysregulation of Axon Guidance Pathways.

The UNC-5 family of netrin receptor genes, predominantly expressed in brain tissues, plays a pivotal role in various neuronal processes. Mutations in genes involved in axon development contribute to a wide spectrum of human diseases, including developmental, neuropsychiatric, and neurodegenerative disorders. The NTN1/DCC signaling pathway, interacting with UNC5C, plays a crucial role in central nervous system axon guidance and has been associated with psychiatric disorders during adolescence in humans. Whole-exome sequencing analysis unveiled two compound heterozygous causative mutations within the UNC5C gene in a patient diagnosed with psychiatric disorders. In silico analysis demonstrated that neither of the observed variants affected the allosteric linkage between UNC5C and NTN1. In fact, these mutations are located within crucial cytoplasmic domains, specifically ZU5 and the region required for the netrin-mediated axon repulsion of neuronal growth cones. These domains play a critical role in forming the supramodular protein structure and directly interact with microtubules, thereby ensuring the functionality of the axon repulsion process. We emphasize that these mutations disrupt the aforementioned processes, thereby associating the UNC5C gene with psychiatric disorders for the first time and expanding the number of genes related to psychiatric disorders. Further research is required to validate the correlation of the UNC5C gene with psychiatric disorders, but we suggest including it in the genetic analysis of patients with psychiatric disorders.

SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Long noncoding RNA UNC5B-AS1 suppresses cell proliferation by sponging miR-24-3p in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities. MATERIALS AND METHODS: GBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The "edger" R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs' target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis. RESULTS: Our results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation. CONCLUSION: The discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.

Identification of UNC5B as a novel aggressive biomarker for osteosarcoma based on basement membrane genes.

BACKGROUND: The prognosis of patients with metastatic osteosarcoma is poor, and the variation of basement membrane genes (BMGs) is associated with cancer metastasis. However, the role of BMGs in osteosarcoma has been poorly studied. METHODS: BMGs were collected and differentially expressed BMGs (DE-BMGs) were found through difference analysis. DE-BMGs were further screened by univariate Cox regression and Lasso regression analyses, and six key BMGs were identified and defined as basement membrane genes signatures (BMGS). Then, BMGS was used to construct the osteosarcoma BMGS risk score system, and the osteosarcoma patients were divided into high- and low-risk groups based on the median risk score. Single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE scores were used to investigate the differences in immune infiltration between the two scoring groups. Additionally, we investigated whether UNC5B affects various features in tumors by bioinformatic analysis and whether UNC5B was involved in multiple biological functions of osteosarcoma cells by wound healing assay, transwell assay, and western blot. RESULTS: The osteosarcoma BMGS risk score reliably predicts the risk of metastasis, patient prognosis, and immunity. UNC5B expression was elevated in osteosarcoma, and correlated with various characteristics such as immune infiltration, prognosis, and drug sensitivity. In vitro assays showed that UNC5B knockdown reduced osteosarcoma cells' capacity for migration and invasion, and EMT process. CONCLUSION: A novel BMGS risk score system that can effectively predict the prognosis of osteosarcoma was developed and validated. The UNC5B gene in this system is one of the key aggressive biomarkers of osteosarcoma.