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.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Stromal netrin 1 coordinates renal arteriogenesis and mural cell differentiation.

The kidney vasculature has a complex architecture that is essential for renal function. The molecular mechanisms that direct development of kidney blood vessels are poorly characterized. We identified a regionally restricted, stroma-derived signaling molecule, netrin 1 (Ntn1), as a regulator of renal vascular patterning in mice. Stromal progenitor (SP)-specific ablation of Ntn1 (Ntn1SPKO) resulted in smaller kidneys with fewer glomeruli, as well as profound defects of the renal artery and transient blood flow disruption. Notably, Ntn1 ablation resulted in loss of arterial vascular smooth muscle cell (vSMC) coverage and in ectopic SMC deposition at the kidney surface. This was accompanied by dramatic reduction of arterial tree branching that perdured postnatally. Transcriptomic analysis of Ntn1SPKO kidneys revealed dysregulation of vSMC differentiation, including downregulation of Klf4, which we find expressed in a subset of SPs. Stromal Klf4 deletion similarly resulted in decreased smooth muscle coverage and arterial branching without, however, the disruption of renal artery patterning and perfusion seen in Ntn1SPKO. These data suggest a stromal Ntn1-Klf4 axis that regulates stromal differentiation and reinforces stromal-derived smooth muscle as a key regulator of renal blood vessel formation.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice.

Purpose: Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. Methods: Aqp5 knockout (Aqp5-/-) and wild-type (Aqp5+/+) mice were studied. LGs were permeabilized and stained with neuronal class III ß-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. Results: Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5-/- mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5-/- mice LGs. Aqp5+/+ mice LG tissue extracts (TEs) promoted Aqp5-/- TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5-/- mice LG TEs inhibited Aqp5+/+ TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. Conclusions: AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5-/- mice LG epithelial cells. This affected sensory and sympathetic nerve fibers' distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.

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.

Relationship between insulin and Netrin-1/DCC guidance cue pathway regulation in the prefrontal cortex of rodents exposed to prenatal dietary restriction.

Fetal restriction (FR) alters insulin sensitivity, but it is unknown how the metabolic profile associated with restriction affects development of the dopamine (DA) system and DA-related behaviors. The Netrin-1/DCC guidance cue system participates in maturation of the mesocorticolimbic DA circuitry. Therefore, our objective was to identify if FR modifies Netrin-1/DCC receptor protein expression in the prefrontal cortex (PFC) at birth and mRNA in adulthood in rodent males. We used cultured HEK293 cells to assess if levels of miR-218, microRNA regulator of DCC, are sensitive to insulin. To assess this, pregnant dams were subjected to a 50% FR diet from gestational day 10 until birth. Medial PFC (mPFC) DCC/Netrin-1 protein expression was measured at P0 at baseline and Dcc/Netrin-1 mRNA levels were quantified in adults 15 min after a saline/insulin injection. miR-218 levels in HEK-293 cells were measured in response to insulin exposure. At P0, Netrin-1 levels are downregulated in FR animals in comparison to controls. In adult rodents, insulin administration results in an increase in Dcc mRNA levels in control but not FR rats. In HEK293 cells, there is a positive correlation between insulin concentration and miR-218 levels. Since miR-218 is a Dcc gene expression regulator and our in vitro results show that insulin regulates miR-218 levels, we suggest that FR-induced changes in insulin sensitivity could be affecting Dcc expression via miR-218, impacting DA system maturation and organization. As fetal adversity is linked to nonadaptive behaviors later in life, this may contribute to early identification of vulnerability to chronic diseases associated with fetal adversity.

Netrin-1 controls inflammation in response to ischemic stroke through altering microglia phenotype.

Introduction: The current approaches that are used to treat ischemic stroke suffer from poor targeting, lack of effectiveness, and potential off-target effects, necessitating the development of new therapeutic strategies to enhance neuronal cell survival and regeneration. This study aimed to investigate the role of microglial Netrin-1 in ischemic stroke, a topic that has not been fully understood. Methods: Netrin-1 levels and its primary receptor expressions were investigated in cerebral microglia from acute ischemic stroke patients and age-matched control subjects. A public database (GEO148350), which supplied RNAseq results for rat cerebral microglia in a middle cerebral artery occlusion (MCAO) model, was analyzed to assess the expression of Netrin-1, its major receptors, and genes related to macrophage function. A microglia-specific gene targeting approach and a delivery system allowing for crossing the blood-brain barrier were applied in a mouse model for ischemic stroke to investigate the role of microglial Netrin-1. Netrin-1 receptor signaling in microglia was observed and the effects on microglial phenotype, apoptosis, and migration were analyzed. Results: Across human patients, rat and mouse models, activation of Netrin-1 receptor signaling was mainly conducted via its receptor UNC5a in microglia, which resulted in a shift in microglial phenotype towards an anti-inflammatory or M2-like state, leading to a reduction in apoptosis and migration of microglia. Netrin-1-induced phenotypic change in microglia exerted protective effects on neuronal cells in vivo during ischemic stroke. Conclusion: Our study highlights the potential of targeting Netrin-1 and its receptors as a promising therapeutic strategy for promoting post-ischemic survival and functional recovery.

Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control.

Mirror movements (MM) disorder is characterized by involuntary movements on one side of the body that mirror intentional movements on the opposite side. We performed genetic characterization of a family with autosomal dominant MM and identified ARHGEF7, a RhoGEF, as a candidate MM gene. We found that Arhgef7 and its partner Git1 bind directly to Dcc. Dcc is the receptor for Netrin-1, an axon guidance cue that attracts commissural axons to the midline, promoting the midline crossing of axon tracts. We show that Arhgef7 and Git1 are required for Netrin-1-mediated axon guidance and act as a multifunctional effector complex. Arhgef7/Git1 activates Rac1 and Cdc42 and inhibits Arf1 downstream of Netrin-1. Furthermore, Arhgef7/Git1, via Arf1, mediates the Netrin-1-induced increase in cell surface Dcc. Mice heterozygous for Arhgef7 have defects in commissural axon trajectories and increased symmetrical paw placements during skilled walking, a MM-like phenotype. Thus, we have delineated how ARHGEF7 mutation causes MM.

Long noncoding RNA DIAPH2-AS1 promotes neural invasion of gastric cancer via stabilizing NSUN2 to enhance the m5C modification of NTN1.

Neural invasion (NI) is a vital pathological characteristic of gastric cancer (GC), which correlates with tumor recurrence and a worse prognosis. Long noncoding RNAs (lncRNAs) play critical roles in various biological processes. However, the involvement of lncRNAs in NI of GC (GC-NI) remains unclear. DIAPH2-AS1 was upregulated in NI-positive GC tissues, which was confirmed by qRT-PCR. The higher expression of DIAPH2-AS1 predicted NI and worse survival for GC patients. Both in vitro and in vivo experiments, including wound-healing assay, Transwell assay, DRG-GC cells co-culture model, the mouse sciatic nerve model, and the lung metastasis model, indicated that DIAPH2-AS1 promoted the migration, invasion, and NI potential of GC cells. Mechanistically, pulldown assay and RNA immunoprecipitation assay revealed that DIAPH2-AS1 interacted with NSUN2. Subsequent experiments indicated that DIAPH2-AS1 stabilized NSUN2 from ubiquitin-proteasomal degradation via masking the K577 and K579 of NSUN2. The protection of DIAPH2-AS1 on NSUN2 improved the stability of NTN1 mRNA via m5C modification, which finally induced GC-NI. Our work uncovered DIAPH2-AS1 as a novel oncogenic lncRNA in GC-NI and validated the DIAPH2-AS1-NSUN2-NTN1 axis as a potential therapeutic target for NI-positive GC.

Restoring bone marrow niche function rejuvenates aged hematopoietic stem cells by reactivating the DNA Damage Response.

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.

Identification of putative regulatory single-nucleotide variants in NTN1 gene associated with NSCL/P.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common polygenetic disease. Although genome-wide association studies (GWAS) identified NTN1 gene as a high-priority candidate of NSCL/P, the comprehensive genetic architecture of NTN1 weren't yet known. Thus, this study aimed to determine full-scale genetic variants of NTN1 for NSCL/P in Chinese Han people. Initially, targeted sequencing of NTN1 gene was performed on 159 NSCL/P patients to identify susceptible single nucleotide polymorphisms (SNPs) associated with NSCL/P. Then, association analysis and burden analysis were separately used to validate the common variants and rare variants identified among large size of samples (1608 NSCL/P cases and 2255 controls). Additionally, NSCL/P subtype association analysis was applied to elucidate the etiology discrepancy of non-syndromic cleft lip with palate (NSCLP) and non-syndromic cleft lip only (NSCLO). Lastly, bioinformatics analysis was performed to annotate and prioritize candidate variants. We found 15 NSCL/P-associated SNPs including rs4791774 (P = 1.10E-08, OR = 1.467, 95% CI: 1.286~1.673) and rs9788972 (P = 1.28E-07, OR = 1.398, 95% CI : 1.235~1.584) originally detected by previous GWASs in Chinese Han ancestry. Four NSCLO risk-associated SNPs and eight specific NSCLP associated SNPs were found. Three SNPs (rs4791331, rs4791774 and rs9900753) were predicted to locate at regulatory region of NTN1. Our study validated the association between NTN1 gene and pathogenesis of NSCL/P and reinforced the hypothesis that NSCLP have a different etiology from NSCLO. We also identified three putative regulatory SNPs in NTN1 gene.

Axon targeting of Drosophila medulla projection neurons requires diffusible Netrin and is coordinated with neuroblast temporal patterning.

How axon guidance pathways are utilized in coordination with temporal and spatial patterning of neural progenitors to regulate neuropil assembly is not well understood. We study this question in the Drosophila medulla using the transmedullary (Tm) projection neurons that target lobula through the inner optic chiasm (IOC). We demonstrate that the Netrin pathway plays multiple roles in guidance of Tm axons and that temporal patterning of medulla neuroblasts determines pioneer versus follower Tm neurons during this process. Loss of Frazzled (Fra) in early-born pioneer Tm neurons leads to IOC defects, while loss of Fra from follower neurons does not affect the IOC. In the follower projection neurons, Fra is required in other targeting steps including lobula branch extension and layer-specific targeting. Furthermore, different from other identified scenarios of Netrin/Fra involved axon guidance in Drosophila, we demonstrate that diffusible Netrin is required for the correct axon targeting and optic lobe organization.

Netrin-1 Monoclonal Antibody-Functionalized Nanoparticle Loaded with Metformin Prevents the Progression of Abdominal Aortic Aneurysms.

Background: Abdominal aortic aneurysms (AAAs) are a global health and economic burden. Therapeutic strategies to inhibit the progression of AAAs are currently lacking. Recently, the therapeutic effect of metformin on aneurysms has attracted considerable interest. However, the unfavorable pharmacokinetic properties of metformin limit its feasibility for AAA treatment. Methods and Results: We constructed a metformin-loaded netrin-1-responsive AAA-targeted nanoparticle (Tgt-NP-Met) for AAA management. Evaluation of the therapeutic effect of Tgt-NP-Met was performed by in vitro and in vivo experiments. Our results showed that the binding of netrin-1 monoclonal antibodies enhanced the AAA-targeting capability of nanoparticles (NPs). Moreover, Tgt-NP-Met administration prevented AAA development and reduced the aneurysm diameter in apolipoprotein E (ApoE)-deficient (ApoE-/-) mice that received continuous infusion of angiotensin II. Furthermore, metformin prevented AAA progression by inhibiting the transformation of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a synthetic phenotype, which is mediated by macrophage infiltration and activation. Conclusion: Our findings identify metformin as a functional suppressor for macrophage-mediated phenotypic transformation of VSMCs and Tgt-NP-Met as an efficient therapeutic strategy for AAA management.

Effects of Netrin-1 and NHE1 Participation on the Migration of Macrophages Driven by CCL2.

This experiment was designed to investigate the relationship between NHE1 gene expression differences between Netrin-1 and NHE1. For this purpose, the blank control, CCL2, CCL2 + Netrin-1 groups were constructed, and cell migration ability was detected by scratch tests and Transwell experiments; Commercial over-expressed NHE1 adenovirus vector (over-expressed NHE1 group), shRNA adenoviral vector silencing NHE1 (silencing NHE1 group) and negative control without carrying virus (negative control group) were subjected to RT-PCR test 24h after infection and pH recovery rate after acid loading was measured. The percentage of wound healing area and the number of cell migration of macrophages in the blank control group, CCL2 group, CCL2+Netrin-1 group, over-expressed NHE1 group, silencing NHE1 group and negative control group were compared. Results showed that in terms of migration ability, the percentage of wound healing area and migration in CCL2 increased (P <0.05), in CCL2 + Netrin-1 (P <0.05) and increased NHE1 mRNA (P <0.05), and not in NHE1 (P <0.05).pH response rate after acid load (NHE1 activity) showed that NHE1 activity was enhanced compared with the blank group, while NHE1 activity in silent NHE1 group decreased (P <0.05); from macrophage migration ability after overexpression/silencing, the percentage of macrophage wound healing area and cell migration increased/decreased compared with CCL2 group and Netrin-1 + CCL2 group (P <0.05). Then Upregulation of NHE1 can promote CCL2-driven macrophage RAW264.7 cell migration, and the downregulation of NHE1 can inhibit its cell migration; Netrin-1 can inhibit CCL2-driven RAW264.7 cell migration regardless of NHE1 regulation.

Csf1r mediates enhancement of intestinal tumorigenesis caused by inactivation of Mir34a.

The CSF1 receptor (CSF1R) encoding mRNA represents a direct target of miR-34a. However, the in vivo relevance of the suppression of CSF1R by miR-34a for intestinal tumor suppression mediated by the p53/miR-34a pathway has remained unknown. Here, Apc Min/+ mice with intestinal-epithelial cell (IEC)-specific deletions of Mir34a showed increased formation of adenomas and decreased survival, whereas deletion of Csf1r decreased adenoma formation and increased survival. In adenomas deletion of Mir34a enhanced proliferation, STAT3 signaling, infiltration with fibroblasts, immune cells and microbes, and tumor stem cell abundance and decreased apoptosis. Deletion of Csf1r had the opposite effects. In addition, homeostasis of intestinal secretory and stem cells, and tumoroid formation were affected in opposite directions by deletion of Mir34a and CSF1R. Concomitant deletion of Csf1r and Mir34a neutralized the effects of the single deletions. mRNAs containing Mir34a seed-matching sites, which encode proteins related to EMT (epithelial-mesenchymal transition), stemness and Wnt signaling, were enriched after Mir34a inactivation in adenomas and derived tumoroids. Netrin-1/Ntn1 and Transgelin/Tagln were characterized as direct targets of Mir34a and Csf1r signaling. Mir34a-inactivation related expression signatures were associated with CMS4/CRISB+D, stage 4 CRCs and poor patient survival. In tumoroids the loss of Mir34a conferred resistance to 5-FU which was mediated by Csf1r. This study provides genetic evidence for a requirement of Mir34a-mediated Csf1r suppression for intestinal stem/secretory cell homeostasis and tumor suppression, and suggests that therapeutic targeting of CSF1R may be effective for the treatment of CRCs with defects in the p53/miR-34a pathway.

Rs9891446 in NTN1 is associated with right-side cleft lip in Han Chinese population.

OBJECTIVES: This study aimed to reveal the association between single-nucleotide polymorphism of NTN1 and subtypes of non-syndromic cleft lip with or without cleft palate (NSCL/P) in Han Chinese Population. DESIGN: Initially, we selected three single-nucleotide polymorphisms (SNP) (rs4791331, rs4791774 and rs9891446) in NTN1 from previous genetic studies. Then we recruited two Han Chinese cohorts (2004 cases and 1823 controls) and divided cases into subgroups: non-syndromic right-side cleft lip, non-syndromic left-side cleft lip, non-syndromic bilateral cleft lip and non-syndromic cleft lip with palate to further evaluate the associations between the subtypes of NSCL/P and SNPs in NTN1. PLINK and Haploview program were utilized to analyze the data. RESULTS: In the association analysis under additive model, we found that G allele at rs9891446 could specifically increase the risk of right-side cleft lip (P = 0.0073, OR = 1.44, 95%CI: 1.1-1.88), which was consistent with the results of association analysis under genotypic model. CONCLUSION: This study showed that rs9891446 of NTN1 was specifically associated with right-side cleft lip in Han Chinese, which indicates that different subtypes of non-syndromic cleft lip have distinct genetic background.

Detection of netrin-1 as a novel biomarker for diagnosis and chemotherapeutic monitoring of lung cancer.

OBJECTIVE: Lung cancer has high morbidity and mortality. We aimed to determine the value of netrin-1 for the diagnosis and chemotherapeutic monitoring of lung cancer. METHODS: Thirty pairs of lung cancer tissues and serum were collected. Netrin-1 expression was detected by immunohistochemistry and enzyme-linked immunosorbent assay. Netrin-1 expression was downregulated in A549 cells using small interfering RNA, and the effect of netrin-1 on cisplatin-induced lung cancer cell apoptosis was determined by flow cytometry. RESULTS: Netrin-1-positivity was significantly higher in lung cancer tissues than in paracarcinoma tissues and high expression of netrin-1 was closely related to a poor prognosis. Serum netrin-1 levels were also significantly higher in lung cancer patients than in healthy donors, and were higher in patients with lung cancer before the beginning of chemotherapy compared with after the completion of four cycles of chemotherapy. Netrin-1 knockdown increased the rate of cisplatin-induced apoptosis in A549 cells. CONCLUSIONS: Netrin-1 expression was increased in tissues and serum from lung cancer patients and decreased after chemotherapy, suggesting that it may be a potential diagnostic marker and indicator of chemosensitivity. Netrin-1 may participate in cisplatin resistance by reducing apoptosis, thus providing a new strategy for addressing chemoresistance in patients with lung cancer.

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.