Deficiency of inflammation-sensing protein neuropilin-2 in myeloid-derived macrophages exacerbates colitis via NF-κB activation.

Neuropilin-2 (NRP2) is a multifunctional protein engaged in the regulation of angiogenesis, lymphangiogenesis, axon guidance, and tumor metastasis, but its function in colitis remains unclear. Here, we found that NRP2 was an inflammation-sensing protein rapidly and dramatically induced in myeloid cells, especially in macrophages, under inflammatory contexts. NRP2 deficiency in myeloid cells exacerbated dextran sulfate sodium salt-induced experimental colitis by promoting polarization of M1 macrophages and colon injury. Mechanistically, NRP2 could be induced via NF-κB activation by TNF-α in macrophages, but exerted an inhibitory effect on NF-κB signaling, forming a negative feedback loop with NF-κB to sense and alleviate inflammation. Deletion of NRP2 in macrophages broke this negative feedback circuit, leading to NF-κB overactivation, inflammatory exacerbation, and more severe colitis. Collectively, these findings reveal inflammation restriction as a role for NRP2 in macrophages under inflammation contexts and suggest that NRP2 in macrophages may relieve inflammation in inflammatory bowel disease. © 2023 The Pathological Society of Great Britain and Ireland.

The SEMA3F-NRP1/NRP2 axis is a key factor in the acquisition of invasive traits in in situ breast ductal carcinoma.

BACKGROUND: A better understanding of ductal carcinoma in situ (DCIS) is urgently needed to identify these preinvasive lesions as distinct clinical entities. Semaphorin 3F (SEMA3F) is a soluble axonal guidance molecule, and its coreceptors Neuropilin 1 (NRP1) and NRP2 are strongly expressed in invasive epithelial BC cells. METHODS: We utilized two cell line models to represent the progression from a healthy state to the mild-aggressive or ductal carcinoma in situ (DCIS) stage and, ultimately, to invasive cell lines. Additionally, we employed in vivo models and conducted analyses on patient databases to ensure the translational relevance of our results. RESULTS: We revealed SEMA3F as a promoter of invasion during the DCIS-to-invasive ductal carcinoma transition in breast cancer (BC) through the action of NRP1 and NRP2. In epithelial cells, SEMA3F activates epithelialmesenchymal transition, whereas it promotes extracellular matrix degradation and basal membrane and myoepithelial cell layer breakdown. CONCLUSIONS: Together with our patient database data, these proof-of-concept results reveal new SEMA3F-mediated mechanisms occurring in the most common preinvasive BC lesion, DCIS, and represent potent and direct activation of its transition to invasion. Moreover, and of clinical and therapeutic relevance, the effects of SEMA3F can be blocked directly through its coreceptors, thus preventing invasion and keeping DCIS lesions in the preinvasive state.

Neuropilin 2 Is a Novel Regulator of Distal Colon Contractility.

Appropriate coordination of smooth muscle contraction and relaxation is essential for normal colonic motility. The impact of perturbed motility ranges from moderate, in conditions such as colitis, to potentially fatal in the case of pseudo-obstruction. The mechanisms underlying aberrant motility and the extent to which they can be targeted pharmacologically are incompletely understood. This study identified colonic smooth muscle as a major site of expression of neuropilin 2 (Nrp2) in mice and humans. Mice with inducible smooth muscle-specific knockout of Nrp2 had an increase in evoked contraction of colonic rings in response to carbachol at 1 and 4 weeks following initiation of deletion. KCl-induced contractions were also increased at 4 weeks. Colonic motility was similarly enhanced, as evidenced by faster bead expulsion in Nrp2-deleted mice versus Nrp2-intact controls. In length-tension analysis of the distal colon, passive tension was similar in Nrp2-deficient and Nrp2-intact mice, but at low strains, active stiffness was greater in Nrp2-deficient animals. Consistent with the findings in conditional Nrp2 mice, Nrp2-null mice showed increased contractility in response to carbachol and KCl. Evaluation of selected proteins implicated in smooth muscle contraction revealed no significant differences in the level of α-smooth muscle actin, myosin light chain, calponin, or RhoA. Together, these findings identify Nrp2 as a novel regulator of colonic contractility that may be targetable in conditions characterized by dysmotility.

CircPI4KA Overexpression Enhances Carcinogenesis and Glycolysis Metabolism in Papillary Thyroid Carcinoma by Causing the miR-1287-5p-Mediated NRP2 Expression Elevation.

Circular RNAs (circRNAs) are implicated in regulating the pathogenesis of papillary thyroid carcinoma (PTC). Herein, we aimed to investigate how circRNA phosphatidylinositol 4-kinase IIIα (circPI4KA, hsa_circ_0062389) functioned as an oncogene in PTC. CircPI4KA, microRNA-1287-5p (miR-1287-5p) and Neuropilin-2 (NRP2) level detection were completed by reverse transcription-quantitative polymerase chain reaction assay. Cell proliferation was assessed through Cell Counting Kit-8 assay, colony formation assay, and EdU assay. Transwell assay was used for detecting migration and invasion abilities. Cell migration was also determined by wound healing assay. Cell apoptosis was assessed using flow cytometry assay. The protein examination was performed using western blot. Glycolysis was evaluated via commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted for target analysis. The role of circPI4KA in vivo was explored and analyzed via tumor xenograft assay. CircPI4KA was significantly upregulated in PTC tissues and cells. Knockdown of circPI4KA suppressed proliferation, migration, invasion, glycolysis, and induced apoptosis of PTC cells. CircPI4KA interacted with miR-1287-5p in PTC cells. The antitumor function of circPI4KA downregulation was reversed by inhibition of miR-1287-5p. The miR-1287-5p directly targeted NRP2, and circPI4KA elevated the NRP2 expression by sponging miR-1287-5p. PTC progression was impeded by miR-1287-5p via targeting NRP2. Silencing circPI4KA inhibited tumor growth in vivo through the miR-1287-5p/NRP2 axis. The collective results revealed that circPI4KA induced the upregulation of NRP2 via sponging miR-1287-5p, thus acting as a carcinogenic factor in PTC.

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

IKKß increases neuropilin-2 and promotes the inhibitory function of CD9+ Bregs to control allergic diseases.

Regulatory B cells (Bregs) potently suppress immune disorders, including allergic contact hypersensitivity (CHS). IKKß overactivation is prominent in various inflammatory diseases. However, its effect on Bregs has not been defined. This study is to investigate the new regulator and inhibitory mechanism of Bregs. IkkßC46A transgenic mice with a Cys46 mutation, resulting in increased IKKß activation, were employed for analysis. IL-10-competent CD9+ Bregs were expanded in IkkßC46A mice and B cell specific-IkkßC46A mutation mice. IkkßC46A mutant CD9+ Bregs had stronger suppressive effects on CD4+ and CD8+ T cells in vitro and CHS responses in vivo. The inhibitory CD9+ Bregs from IkkßC46A mice were characterized by upregulated Neuropilin 2 (Nrp2) and IL-10 in comparison with that of Ikkßwt mice. Interestingly, increased expression of Nrp2 was observed in CD9+ Bregs compared with that of CD9- B cells in wild-type mice. The suppressive activity of wild-type CD9+ Bregs in vitro was attenuated by inhibition of Nrp2 on Bregs or silencing its ligand Sema3f on CD4+ T cells. Our findings delineate a distinct role of IKKß activation in enhancing Bregs to disturb the immune balance. It identifies Nrp2 as a novel regulatory molecule of Bregs that partly contributes to B cell-mediated immune tolerance.

A Novel circUBR4/miR-491-5p/NRP2 ceRNA Network Regulates Oxidized Low-density Lipoprotein-induced Proliferation and Migration in Vascular Smooth Muscle Cells.

ABSTRACT: Vascular smooth muscle cells (VSMCs) play critical roles in the progression of atherosclerosis. Circular RNA (circRNA) ubiquitin protein ligase E3 component n-recognin 4 (circUBR4) has been shown to regulate VSMC migration and proliferation. In this study, we sought to identify the mechanism in the regulation of circUBR4. CircUBR4, microRNA (miR)-491-5p, and Neuropilin-2 (NRP2) were quantified by quantitative real-time polymerase chain reaction (PCR) and western blot. Cell proliferation was evaluated by Cell Counting Kit-8 and 5-Ethynyl-2'-Deoxyuridine assays. Cell migration was examined by wound-healing and transwell invasion assays. The direct relationship between miR-491-5p and circUBR4 or NRP2 was validated by dual-luciferase reporter and RNA immunoprecipitation assays. Our data indicated that in VSMCs, ox-LDL induced circUBR4 expression. Silencing endogenous circUBR4 attenuated VSMC proliferation and migration induced by ox-LDL. Mechanistically, circUBR4 targeted miR-491-5p by pairing to miR-491-5p. Moreover, miR-491-5p was identified as a downstream mediator of circUBR4 function in ox-LDL-treated VSMCs. NRP2 was a direct target of miR-491-5p, and circUBR4 acted as a competing endogenous RNA for miR-491-5p to regulate NRP2 expression. In addition, NRP2 was a functionally downstream effector of miR-491-5p in regulating ox-LDL-evoked VSMC proliferation and migration. Our findings identify a new competing endogenous RNA network, the circUBR4/miR-491-5p/NRP2 axis, for the regulation of circUBR4 in VSMC migration and proliferation.

Circ_CHFR Promotes Platelet-Derived Growth Factor-BB-Induced Proliferation, Invasion, and Migration in Vascular Smooth Muscle Cells via the miR-149-5p/NRP2 Axis.

ABSTRACT: Circular RNA checkpoint with forkhead and ring finger domains (circ_CHFR) were reported to regulate vascular smooth muscle cell (VSMC) dysfunction during atherosclerosis (AS). However, the molecule mechanism of circ_CHFR in AS remains largely unclear. Human VSMCs (HVSMCs) were exposed to platelet-derived growth factor-BB (PDGF-BB) in vitro. Levels of circ_CHFR, microRNA (miR)-149-5p, and neuropilin 2 (NRP2) were determined using quantitative real-time polymerase chain reaction and western blot. Cell proliferation, migration, and invasion were analyzed using cell counting kit-8, colony formation, flow cytometry, wound healing, and transwell assays. The binding interaction between miR-149-5p and circ_CHFR or NRP2 was investigated using the dual-luciferase reporter and RNA immunoprecipitation assays. Circ_CHFR was elevated in PDGF-BB-induced HVSMCs in a dose-independent manner. Silencing of circ_CHFR reversed PDGF-BB-evoked promotion of cell proliferation, migration and invasion, as well as suppression of cell apoptosis in HVSMCs. Mechanistically, circ_CHFR directly bound to miR-149-5p, and miR-149-5p inhibition attenuated the effects of circ_CHFR knockdown on PDGF-BB-induced HVSMCs. Besides, NRP2 was confirmed to be a target of miR-149-5p, and circ_CHFR could regulate NRP2 expression through sponging miR-149-5p. Moreover, miR-149-5p overexpression abolished PDGF-BB-triggered enhancement of cell proliferation, migration, and invasion by targeting NRP2. Circ_CHFR promoted the proliferation, invasion, and migration of PDGF-BB-induced HVSMCs through miR-149-5p/NRP2 axis, providing a new insight into the pathogenesis of AS and a potential therapeutic target for AS treatment.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

Neuropilin (NRPs) Related Pathological Conditions and Their Modulators.

Neuropilin 1 (NRP1) represents one of the two homologous neuropilins (NRP, splice variants of neuropilin 2 are the other) found in all vertebrates. It forms a transmembrane glycoprotein distributed in many human body tissues as a (co)receptor for a variety of different ligands. In addition to its physiological role, it is also associated with various pathological conditions. Recently, NRP1 has been discovered as a coreceptor for the SARS-CoV-2 viral entry, along with ACE2, and has thus become one of the COVID-19 research foci. However, in addition to COVID-19, the current review also summarises its other pathological roles and its involvement in clinical diseases like cancer and neuropathic pain. We also discuss the diversity of native NRP ligands and perform a joint analysis. Last but not least, we review the therapeutic roles of NRP1 and introduce a series of NRP1 modulators, which are typical peptidomimetics or other small molecule antagonists, to provide the medicinal chemistry community with a state-of-the-art overview of neuropilin modulator design and NRP1 druggability assessment.

Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors.

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 µg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 µg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner.IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

Circ-LDLRAD3 Enhances Cell Growth, Migration, and Invasion and Inhibits Apoptosis by Regulating MiR-224-5p/NRP2 Axis in Gastric Cancer.

BACKGROUND: Emerging as a newly discovered type of noncoding RNAs, circular RNAs have been manifested as a crucial regulator in tumorigenesis of human malignancies, including gastric cancer (GC). Although circ-LDLRAD3 has been revealed as an oncogene in pancreatic cancer, the underlying role of circ-LDLRAD3 in GC remains poorly understood. AIMS: Exploring the underlying function of circ-LDLRAD3 on GC progression. METHODS: Circ-LDLRAD3 expression was detected through RT-qPCR. EdU, colony formation, TUNEL, and transwell assays were performed to analyze the function of circ-LDLRAD3 on GC progression. Luciferase reporter and RIP assays were applied to testify the interaction between circ-LDLRAD, miR-224-5p, and NRP2 in GC. RESULTS: We detected preliminarily the expression of circ-LDLRAD3 and observed a markedly high expression of circ-LDLRAD3 in GC cells. Besides, circ-LDLRAD3 was featured with loop structure. Biological function assays testified that silenced circ-LDLRAD3 inhibited cell proliferation, migration, and invasion capacity but facilitated apoptosis of GC cells. Molecular mechanism assays uncovered that circ-LDLRAD3 combined with miR-224-5p in GC. Moreover, rescue assays delineated that inhibited expression of miR-224-5p could restore the inhibitive influence of circ-LDLRAD3 knockdown on the progression of GC. Moreover, neuropilin 2 (NRP2) was a downstream target of miR-224-5p. Additionally, circ-LDLRAD3 regulated NRP2 expression by sponging miR-224-5p in GC. Furthermore, circ-LDLRAD3 depletion-mediated effect on GC progression could be reversed by overexpressing NRP2. CONCLUSIONS: Circ-LDLRAD3 facilitates GC progression by regulating miR-224-5p/NRP2 axis, providing new insights for the researches of GC treatment.

Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.

A radial axis defined by semaphorin-to-neuropilin signaling controls pancreatic islet morphogenesis.

The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered. Here, we identify a radial signaling axis that instructs developing islet cells to disperse throughout the pancreas. A screen of pancreatic extracellular signals identified factors that stimulated islet cell development. These included semaphorin 3a, a guidance cue in neural development without known functions in the pancreas. In the fetal pancreas, peripheral mesenchymal cells expressed Sema3a, while central nascent islet cells produced the semaphorin receptor neuropilin 2 (Nrp2). Nrp2 mutant islet cells developed in proper numbers, but had defects in migration and were unresponsive to purified Sema3a. Mutant Nrp2 islets aggregated centrally and failed to disperse radially. Thus, Sema3a-Nrp2 signaling along an unrecognized pancreatic developmental axis constitutes a chemoattractant system essential for generating the hallmark morphogenetic properties of pancreatic islets. Unexpectedly, Sema3a- and Nrp2-mediated control of islet morphogenesis is strikingly homologous to mechanisms that regulate radial neuronal migration and cortical lamination in the developing mammalian brain.

Neuropilin-2 contributes to tumor progression in preclinical models of small intestinal neuroendocrine tumors.

The identification of novel regulators of tumor progression is a key challenge to gain knowledge on the biology of small intestinal neuroendocrine tumors (SI-NETs). We recently identified the loss of the axon guidance protein semaphorin 3F as a protumoral event in SI-NETs. Interestingly the expression of its receptor neuropilin-2 (NRP-2) was still maintained. This study aimed at deciphering the potential role of NRP-2 as a contributor to SI-NET progression. The role of NRP-2 in SI-NET progression was addressed using an approach integrating human tissue and serum samples, cell lines and in vivo models. Data obtained from human SI-NET tissues showed that membranous NRP-2 expression is present in a majority of tumors, and is correlated with invasion, metastatic abilities, and neovascularization. In addition, NRP-2 soluble isoform was found elevated in serum samples from metastatic patients. In preclinical mouse models of NET progression, NRP-2 silencing led to a sustained antitumor effect, partly driven by the downregulation of VEGFR2. In contrast, its ectopic expression conferred a gain of aggressiveness, driven by the activation of various oncogenic signaling pathways. Lastly, NRP-2 inhibition led to a decrease of tumor cell viability, and sensitized to therapeutic agents. Overall, our results point out NRP-2 as a potential therapeutic target for SI-NETs, and will foster the development of innovative strategies targeting this receptor. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical significance of neuropilin-2 expression in laryngeal squamous cell carcinoma.

PURPOSE: The purpose of the study is to evaluate the expression of NRP-2 and explore its role in Laryngeal squamous cell carcinoma (LSCC). MATERIALS AND METHODS: NRP-2 expression in 70 primary LSCC tissue specimens were analyzed by immunohistochemistry and correlated with clinicopathological parameters and patients´ survival rate. Additionally, 9 paired LSCC tissues were evaluated for NRP-2 expression by Western blotting. RESULTS: The Western blotting indicated that NRP-2 expression levels in LSCC were significantly higher than those in the paraneoplastic tissues (P < 0.05). Immunohistochemistry staining revealed that NRP-2 was detected in all primary tumor samples, moreover, high expression of NRP-2 was significantly correlated with TNM stage (P < 0.05), clinical stage (P < 0.05), histological classification (P < 0.05), lymph node metastasis (P < 0.05) and recurrence (P = 0.001). Survival curves determined by the Kaplan-Meier method showed that high expression of NRP-2 can reduce overall survival (both group P < 0.05). Then we combined the NRP-2 expression and lymph node status, and Kaplan-Meier survival showed patients with high expression of NRP-2 or lymph node metastasis (+) had both shorter disease-free and overall survival than others (both P < 0.05). Multivariate Cox proportional hazards model analysis confirmed that histological grade (P = 0.045), lymph node metastasis (P = 0.020) and high expression of NRP-2 (P = 0.033) were statistically significant, independent predictor of prognosis. CONCLUSIONS: NRP-2 may contribute to LSCC progression and represents as a novel prognostic indicator as well as a potential therapeutic target for LSCC.

[Effect of RNA interference targeting neuropilin-2 gene on proliferation and apoptosis of colon cancer cell line HCT-8].

Objective: To investigate the effects of down-regulation of expression of neuropilin-2 (NRP-2) by RNA interference (RNAi) technique on proliferation and apoptosis of HCT-8 colon cancer cells. Methods: NRP2-siRNA and negative control (NControl)-siRNA were transferred into HCT-8 colon cancer cells by liposomes (lip2000) as transfection group and negative control group, and phosphate buffered solution (PBS) was added as blank control group. Quantitative reverse transcription PCR (RT-qPCR) and Western blot were used to detect the transfection effect. The proliferation of cells in the three groups was examined by cell counting kit (CCK) assay, colony-forming unit assay and Ki-67 protein staining assay, respectively. Moreover, the apoptosis of cells in the three groups was determined by acridine orange/propranidine iodide (AO/PI) staining method. Results: The results of RT-qPCR and Western blot showed that the relative expression of NRP-2 mRNA and the content of NRP-2 protein in the transfer group decreased (0.46±0.05 vs 0.99±0.05 and 1.00±0.06; 1.04±0.06 vs 1.73±0.09 and 1.65±0.11) (all P<0.05). The results of CCK-8 demonstrated that the optical density of transfection group was significantly lower than that of the negative control group and the blank control group(24 h: 0.53±0.04 vs 0.82±0.07 and 0.87±0.07; 48 h: 0.54±0.05 vs 1.00±0.09 and 1.17±0.05; 72 h: 0.75±0.05 vs 1.31±0.13 and 1.50±0.03; 96 h:1.05±0.04 vs 1.46±0.09 and 1.86±0.06) (all P<0.05). The results of colony-forming unit assay indicated that the proliferation ability of the cells in the transfer group was significantly lower than that in the other two groups (134.67±8.74 vs 245.33±19.14 and 300.33±14.01, P<0.05). The results of Ki-67 protein staining assay showed that compared with the negative control group and blank control group, the expression of Ki-67 protein was significantly decreased in the transfection group (5.93±0.22 vs 8.36±0.09 and 8.70±0.21, P<0.05). The results of AO/PI assay revealed that the ratio of apoptotic cells to living cells in the transfer group was significantly higher than that in the other two groups (0.43±0.07 vs 0.14±0.04 and 0.11±0.04, P<0.05). Conclusion: The proliferation ability of HCT-8 colon cancer cells decreases, and the apoptosis ability increases by decreasing the expression of NRP-2.

LncRNA XIST facilitates proliferation and epithelial-mesenchymal transition of colorectal cancer cells through targeting miR-486-5p and promoting neuropilin-2.

This study was designed to acertain whether the long noncoding RNA (lncRNA) X-inactive specific transcript (XIST)/miR-486-5p/neuropilin-2 (NRP-2) pathway might promote the viability and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells. In this investigation, we included 317 pathologically confirmed CRC patients and purchased several human CRC cells (i.e. HCT116, HT29, SW620, and SW480). Moreover, pcDNA3.1-XIST, si-XIST, miR-486-5p mimic, miR-486-5p inhibitor, and pcDNA3.1-NRP-2 were transfected into the CRC cells. And the dual-luciferase reporter gene assay managed to verify the targeted relationships among XIST, miR-486-5p, and NRP-2. Ultimately, the MTT assay, flow cytometry, colony formation assay, and transwell assay were carried out to assess the influence of XIST, miR-486-5p, and NRP-2 on the proliferation, apoptosis, migration, and invasion of CRC cells. Our study results demonstrated that CRC tissues and cells were detected with significantly elevated XIST and NRP-2 expressions as well as markedly reduced miR-486-5p expression when compared with normal tissues and cells (all p < 0.05). Besides this, the highly expressed XIST and NRP-2, as well as the lowly expressed miR-486-5p all could substantially encourage proliferation and EMT of CRC cells and simultaneously restrict apoptosis of the cells ( p < 0.05). Moreover, XIST was found to directly target miR-486-5p, and NRP-2 was directly targeted and modulated by miR-486-5p. Finally, CRC cells of the miR-NC + pcDNA3.1-NRP-2 groups showed stronger proliferation, viability, and EMT than those of miR-NC and miR-486-5p mimic groups ( p < 0.05). In conclusion, the XIST/miR-486 -5p/NRP-2 axis appeared to participate in the progression of CRC, which could assist in developing efficacious therapies for CRC.

Attractant and repellent cues cooperate in guiding a subset of olfactory sensory axons to a well-defined protoglomerular target.

Olfactory sensory axons target well-defined intermediate targets in the zebrafish olfactory bulb called protoglomeruli well before they form odorant receptor-specific glomeruli. A subset of olfactory sensory neurons are labeled by expression of the or111-7:IRES:GAL4 transgene whose axons terminate in the central zone (CZ) protoglomerulus. Previous work has shown that some of these axons misproject to the more dorsal and anterior dorsal zone (DZ) protoglomerulus in the absence of Netrin 1/Dcc signaling. In search of additional cues that guide these axons to the CZ, we found that Semaphorin 3D (Sema3D) is expressed in the anterior bulb and acts as a repellent that pushes them towards the CZ. Further analysis indicates that Sema3D signaling is mediated through Nrp1a, while Nrp2b also promotes CZ targeting but in a Sema3D-independent manner. nrp1a, nrp2b and dcc transcripts are detected in or111-7 transgene-expressing neurons early in development and both Nrp1a and Dcc act cell-autonomously in sensory neurons to promote accurate targeting to the CZ. dcc and nrp1a double mutants have significantly more DZ misprojections than either single mutant, suggesting that the two signaling systems act independently and in parallel to direct a specific subset of sensory axons to their initial protoglomerular target.