Slit2 mRNA Research Articles

METTL3-Dependent N6-Methyladenosine Modification Programs Human Neural Progenitor Cell Proliferation.

METTL3, a methyltransferase responsible for N6-methyladenosine (m6A) modification, plays key regulatory roles in mammal central neural system (CNS) development. However, the specific epigenetic mechanisms governing human CNS development remain poorly elucidated. Here, we generated small-molecule-assisted shut-off (SMASh)-tagged hESC lines to reduce METTL3 protein levels, and found that METTL3 is not required for human neural progenitor cell (hNPC) formation and neuron differentiation. However, METTL3 deficiency inhibited hNPC proliferation by reducing SLIT2 expression. Mechanistic studies revealed that METTL3 degradation in hNPCs significantly decreased the enrichment of m6A in SLIT2 mRNA, consequently reducing its expression. Our findings reveal a novel functional target (SLIT2) for METTL3 in hNPCs and contribute to a better understanding of m6A-dependent mechanisms in hNPC proliferation.

Inhibition of miR-200b-3p alleviates hypoxia-ischemic brain damage via targeting Slit2 in neonatal rats

BackgroundBrain damage in premature infants often occurs in very low birth weight infants (VLBW) as a result of hypoxia-ischemia and can lead to cognitive impairment and movement disorders. Many miRNAs have been demonstrated to participate in hypoxia-ischemic brain damage (HIBD). This study was designed to investigate the roles of miR-200b-3p in brain damage of neonatal rats induced by hypoxia-ischemia. Methods and resultsThree-day-old SD rats were used to establish the model of hypoxia-ischemic brain injury mimicking premature infants. RT-qPCR showed that miR-200b-3p was up-regulated in rat brains at the early stage following hypoxia-ischemic treatment. Bioinformatics analysis identified that Slit2 is a target gene of miR-200b-3p and luciferase reporter gene assay confirmed that miR-200b-3p can interact with and target Slit2 mRNA. Inhibition of miR-200b-3p by antagomir increased Slit2 expression at both the mRNA and protein levels in rat brains. TUNEL assay and transmission electron microscopy (TEM) analysis showed decreased numbers of apoptotic neurons in the hypoxia-ischemia-treated animals as a result of administration of miR-200b-3p antagomir. Administration of miR-200b-3p antagomir attenuated spatial and learning memory loss in the animals induced by hypoxia-ischemia as compared to controls. ConclusionOur study has demonstrated that Slit2 is a target gene of miR-200b-3p and that the hypoxia-ischemic brain damage in neonatal rats was alleviated by inhibiting miR-200b-3p via Slit2. miR-200b-3p may be a potential therapeutic target of HIBD for further investigation.

TGF‐β1‐induced miR‐424 Mediates Pulmonary Myofibroblast Differentiation via Targeting Slit2

Background and objectiveIdiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease. Transforming growth factor (TGF)‐β1, a major profibrogenic cytokine, induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of extracellular matrix, leading to loss of lung elasticity and function. Emerging evidence has implicated the importance of microRNAs (miRNAs) in the development of IPF but their regulation and individual pathological roles remain largely unknown. The objective of our study was to define the pro‐fibrotic roles and mechanisms of miRNAs in TGF‐β1‐induced pulmonary fibroblast differentiation.MethodsWe used both RNA sequencing and quantitative RT‐PCR strategies to systematically study TGF‐β1‐induced alternations of miRNAs in human lung fibroblasts (HLF). Significantly upregulated miRNAs in HLF were confirmed in lung tissue of IPF patients. Then, we overexpressed the identified miRNAs or their inhibitors to determine their importance in TGF‐β1‐induced HLF differentiation. Western blot and immunofluorescence staining were used to measure the changes in TGF‐β1 signaling and HLF differentiation. Finally, we searched for the putative targets of these miRNAs using online algorithms (Targetscan and mirSVR) and luciferase reporter assays were performed to determine whether the predicted binding site is directly targeted.ResultsWe identified miR‐424 as an important TGF‐β1‐induced miRNA in HLF. Quantitative RT‐PCR confirmed that miR‐424 expression was increased by 2.5‐fold in HLF in response to TGF‐β1 and was 2‐fold higher in IPF patients as compared to normal subjects. Overexpression of a miR‐424 inhibitor in HLF reduced TGF‐β1‐induced expression of classic myofibroblast differentiation markers including α‐smooth muscle actin (α‐SMA) and connective tissue growth factor (CTGF), whereas a miR‐424 mimic significantly enhanced TGF‐β1‐induced differentiation. Interestingly, upregulation of miR‐424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of the canonical TGF‐β1 signaling pathway. In addition, TGF‐β1‐induced Smad3 phosphorylation in HLF was reduced by the miR‐424 inhibitor. Among the putative targets of miR‐424, Slit2 inhibits TGF‐β1‐induced Smad3 phosphorylation. Luciferase reporter assays showed that miR‐424 represses wild‐type Slit2‐3′UTR reporter activity without effects on a mutant Slit2‐3′UTR reporter, indicating the direct regulation by miR‐424 of the 3′UTR of Slit2 mRNA. Indeed, overexpression of the miR‐424 mimic down‐regulated Slit2 expression in HLF, consistent with the reduced Slit2 expression in IPF patients.ConclusionMiR‐424 plays a pro‐fibrotic role in TGF‐β1‐induced HLF differentiation. It functions as a positive feed‐back regulator of the TGF‐β1 signaling pathway via targeting Slit2. Thus, inhibition of miR‐424 may provide a new therapeutic strategy to prevent IPF progression.Support or Funding InformationSupported by grants from NIH R01HL116849 to YT and AG053553 to KB, Nebraska State LB595 to YT and PWAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low Expression and Promoter Hypermethylation of the Tumour Suppressor SLIT2, are Associated with Adverse Patient Outcomes in Diffuse Large B Cell Lymphoma.

SLIT2 has been classified as a major tumour suppressor gene due to its frequent inactivation in different cancer types. However, alterations of SLIT2 expression and relation to patient outcomes in diffuse large B cell lymphoma (DLBCL) remain undefined. The aim of this study was to investigate the expression and the methylation status of SLIT2 gene as well as its relation to patient outcomes in DLBCL. Immunohistochemical (IHC) staining was carried out to detect the expression of SLIT2 in a series of 108 DLBCL cases. Re-analysis of previously published dataset (GSE10846) that measured gene expression in DLBCL patients who had received CHOP or R-CHOP therapy was performed to identify associations between SLIT2 and patients survival. Laser capture microdissection was performed to isolate GC B cells and DLBCL primary tumor cells. Bisulfite treatment and methylation-specific PCR (MSP) analysis were done to assess SLIT2 promotor methylation status. We report that the expression of SLIT2 protein was reduced in a subset of DLBCL cases and this was significantly correlated with advanced clinical stage (p = 0.041) and was an independent predictor of worse overall survival (OS) (p = 0.012). Re-analysis of published gene expression data showed that reduced SLIT2 mRNA expression was significantly correlated with worse OS in R-CHOP-treated ABC DLBCL patients (p = <0.01). Hypermethylation of the SLIT2 promotor was significantly correlated with low SLIT2 expression (p = 0.009). Our results provide a novel evidence of reduced expression of SLIT2 that is associated with promoter hypermethylation and adverse outcomes in patients with DLBCL.

Slit2 Modulates the Inflammatory Phenotype of Orbit-Infiltrating Fibrocytes in Graves' Disease.

Human CD34+ fibrocytes, circulating monocyte lineage progenitor cells, have recently been implicated in thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves' disease (GD). Fibrocytes express constitutive MHC class II (MHC-2) and, surprisingly, thyroglobulin (Tg) and functional thyrotropin (TSH) receptor (TSHR). Underlying expression of these thyroid proteins is the autoimmune regulator protein (AIRE). Fibrocytes respond robustly to TSH and thyroid-stimulating Igs by generating extremely high levels of inflammatory cytokines, such as IL-6. In TAO, they appear to infiltrate the orbit, where they transition to CD34+ orbital fibroblasts (OF). There, they coexist with CD34- OF as a mixed fibroblast population (GD-OF). In contrast to fibrocytes, GD-OF express vanishingly low levels of MHC-2, Tg, TSHR, and AIRE. Further, the amplitude of IL-6 induction by TSH in GD-OF is substantially lower. The molecular basis for this divergence between fibrocytes and CD34+ OF remains uncertain. In this article, we report that Slit2, an axon guidance glycoprotein, is constitutively expressed by the CD34- OF subset of GD-OF. Culture conditioned medium (CM) generated by incubating with GD-OF and CD34- OF substantially reduces levels of MHC-2, Tg, TSHR, and AIRE in fibrocytes. Expression can be restored by specifically depleting CM of Slit2. The effects of CD34- OF CM are mimicked by recombinant human Slit2. TSH induces Slit2 levels in GD-OF by enhancing both Slit2 gene transcription and mRNA stability. These findings suggest that Slit2 represents a TSH-inducible factor within the TAO orbit that can modulate the inflammatory phenotype of CD34+ OF and therefore may determine the activity and severity of the disease.

1. Targeting Slit2-Robo Signaling as a Novel Therapy for Diabetic Nephropathy

Diabetes is a chronic disease that can lead to life-threatening complications such as diabetic nephropathy (DN). An early manifestation of DN is hyperfiltration, a predictor of poor renal outcomes. Hyperfiltration is partly driven by capillary growth (angiogenesis) within the glomeruli, the filtration units of the kidney. A major stimulus for diabetic glomerular angiogenesis is vascular endothelial growth factor (VEGF). VEGF neutralization blocks hyperfiltration in diabetes, but VEGF blockade itself can lead to renal injury. Endothelial cells express Robo1 and Robo4, which serve as receptors for the secreted ligand Slit2. Robo4 activation by Slit2 inhibits endothelial angiogenesis, whereas Slit2 activation of Robo1 promotes angiogenesis.In search of novel therapies for diabetes-induced glomerular hyperfiltration, we hypothesized that diabetes affects the expression of Slit2 and/or its Robo receptors. We further tested whether Slit2 administration attenuates VEGF-driven diabetic capillary growth.Diabetes was induced by injection of streptozocin (STZ) in male rats. Age- and gender-matched non-diabetic rats were followed as controls. 3 weeks later, kidneys were harvested from both groups, and the expression of Slit2, Robo1 and Robo4 analyzed. In parallel, male diabetic rats were randomized to receive Slit2 or saline injections every 3 days. After 3 weeks, kidney filtration and capillary growth were measured in both groups.Diabetes was associated with a reduction in kidney Robo4 expression, whereas Slit2 and Robo1 mRNA levels were unchanged. Slit2 administration attenuated the diabetes-induced rise in kidney filtration and glomerular capillary growth. Our results suggest that targeting Slit2-Robo signalling may have therapeutic importance for early diabetic nephropathy.

The expression of the Slit-Robo signal in the retina of diabetic rats and the vitreous or fibrovascular retinal membranes of patients with proliferative diabetic retinopathy.

PurposeThe Slit-Robo signal has an important role in vasculogenesis and angiogenesis. Our study examined the expression of Slit2 and its receptor, Robo1, in a rat model of streptozotocin-induced diabetes and in patients with proliferative diabetic retinopathy.MethodsDiabetes was induced in male Sprague-Dawley rats via a single, intraperitoneal injection of streptozotocin. The rats were sacrificed 1, 3 or 6 months after the injection. The expression of Slit2 and Robo1 in retinal tissue was measured by real-time reverse transcription polymerase chain reaction (RT-PCR), and protein levels were measured by western blotting and immunohistochemistry. Recombinant N-Slit2 protein was used to study the effects of Slit2 on the expression of VEGF in vivo. The concentration of Slit2 protein in human eyes was measured by enzyme-linked immunosorbent assay in 27 eyes with proliferative diabetic retinopathy and 28 eyes in control group. The expression of Slit2, Robo1 and VEGF in the excised human fibrovascular membranes was examined by fluorescence immunostaining and semi-quantitative RT-PCR.ResultsThe expression of Slit2 and Robo1 in the retina was altered after STZ injection. Recombinant N-Slit2 protein did not increase the retinal VEGF expression. Vitreous concentrations of Slit2 were significantly higher in the study group than in the control group. In the human fibrovascular membranes of the study group, the co-localization of VEGF with the markers for Slit2 and Robo1was observed. The expression of Slit2 mRNA, Robo1 mRNA, and VEGF mRNA was significantly higher in human fibrovascular proliferative diabetic retinopathy membranes than in the control membranes.ConclusionsThe alteration of Slit2 and Robo1 expression in the retinas of diabetic rats and patients with proliferative diabetic retinopathy suggests a role for the Slit-Robo signal in the various stages diabetic retinopathy. Further studies should address the possible involvement of the Slit-Robo signal in the pathophysiological progress of diabetic retinopathy.

The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea.

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.

Slit2 and Robo1 expression as biomarkers for assessing prognosis in brain glioma patients

ObjectivesThis study was conducted to investigate the clinical significance of Slit2 and Robo1 expression in prognosis of patients with brain gliomas. MethodsHuman brain tissue samples were collected from normal glial tissues (control), low- and high-grade glioma tissues. Slit2 and Robo1 expression levels in cells were assessed by an immunohistochemistry (IHC), and population of the Slit2- and Robo1-presenting patients was examined. The Slit2 and Robo1 mRNA expression levels in three types of the brain cells was determined by RT-PCR. ResultsSlit2+ cell counts were decreased with increased Robo1+ cells in the low-grade and high-grade glioma tissues as compared to the control. The percentage of cells expressing Slit2 decreased from the control to the high-grade glioma and the percentage of cells expressing Robo1 in low- and high-grade gliomas was increased as compared to the control (P < 0.01). The decrease in the Slit2 mRNA expression was associated with the increase in the Robo1 mRNA expression in the low- and high-grade gliomas (P < 0.01 or 0.05). Survival time for patients with Slit2-/Robo1+ gliomas was shorter than patients with Slit2+/Robo1+ gliomas in the investigated cohorts (P < 0.01). ConclusionSlit2 and Robo1 expression levels serve as a biomarker with utility in grading gliomas as well as predicting patient survival. The change in Slit2 expression is more reliable and effective than Robo1 expression in predicting a poor prognosis of brain glioma patients.

Slit and Semaphorin signaling governed by Islet transcription factors positions motor neuron somata within the neural tube

Motor neurons send out axons to peripheral muscles while their cell bodies remain in the ventral spinal cord. The unique configuration of motor neurons spanning the border between the CNS and PNS has been explained by structural barriers such as boundary cap (BC) cells, basal lamina and radial glia. However, mechanisms in motor neurons that retain their position have not been addressed yet. Here we demonstrate that the Islet1 (Isl1) and Islet2 (Isl2) transcription factors, which are essential for acquisition of motor neuron identity, also contribute to restrict motor neurons within the neural tube. In mice that lack both Isl1 and Isl2, large numbers of motor neurons exited the neural tube, even prior to the appearance of BC cells at the ventral exit points. Transcriptional profiling of motor neurons derived from Isl1 null embryonic stem cells revealed that transcripts of major genes involved in repulsive mechanisms were misregulated. Particularly, expression of Neuropilin1 (Npr1) and Slit2 mRNA was diminished in Islet mutant mice, and these could be target genes of the Islet proteins. Consistent with this mechanism, Robo and Slit mutations in mice and knockdown of Npr1 and Slit2 in chick embryos caused motor neurons to migrate to the periphery. Together, our study suggests that Islet genes engage Robo-Slit and Neuropilin–Semaphorin signaling in motor neurons to retain motor somata within the CNS.

P18 Robo1/4-slit2 Expression In Pulmonary Vascular Cells: Implications For Pah?

Introduction and objectives Pulmonary artery hypertension (PAH) is associated with inappropriate vascular remodelling and inflammation. Recent studies have shown that vascular cells express the transmembrane roundabout (Robo) proteins, Robo1 and Robo4, and that interaction with a secreted glycoprotein ligand, Slit2, controls cell migratory and inflammatory response. We hypothesise that Robo1, Robo4 and Slit2 are expressed on pulmonary artery (PA) endothelial cells (EC) and smooth muscle cells (SMC). We also hypothesise that Slit2 will modulate PAEC and PASMC migration and inflammatory mediator release. Methods Real-time-PCR determination of Robo1, Robo4 and Slit2 expression and the house-keeping gene, GAPDH, in PAEC, PASMC and for comparison, human umbilical vein endothelial cells (HUVEC); following incubation with TNFα (10ng/ml) or Slit2N (10nM) for 2h. Enzyme-linked immunosorbent assay measurements of granulocyte-macrophage-colony stimulating factor (GM-CSF) in supernatants of HUVEC, PAEC or PASMC pre-treated (1h) with Slit2N, followed by TNFα (17h). Migration assays (PAEC or PASMC) towards serum-containing medium (0.05 and 0.02%, respectively), for 4h with/ without Slit2N. Results Basal mRNA expression of Robo1, Robo4 and Slit2 was detected in PAEC, PASMC and HUVEC (n = 3–4). Slit2N (2h) significantly (p Conclusion The novel discovery of Robo1, Robo4 and Slit2 mRNA in PAEC and PASMC; and that Slit2 down-regulated Robo4 and Slit2 in PAEC, but not PASMC/HUVEC, might suggest negative feedback on the Robo4-Slit2 axis unique to PAEC. That neither PAEC nor PASMC responded to Slit2 in functional assays could reflect limitations in experimental assays. However, down-regulation of Robo4-Slit2 in PAEC might also explain lack of effect on GM-CSF release, when compared with HUVEC. Further studies to better delineate the role of the Robo-Slit2 pathway in PAH are required.

Abstract 127: Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia

Abstract Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in tumor invasion. The SLIT2/mir-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of directional migration and is epigenetically silenced in many cancers. We propose that mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma and inhibits tumor dissemination following the conversion of precursor lesions to invasive carcinoma. We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 mRNA while KRAS-independent lines show silenced SLIT2 expression. Using chromatin immunoprecipitation (ChIP) and Sequenom, we confirmed that loss of SLIT2 in KRAS-independent lines was due to enrichment of silencing histone mark H3K27me3 and DNA hypermethylation at the SLIT2 promoter, respectively. Activating marks H3K4me2 and H4ac are found at the SLIT2 promoter in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression in pancreatic cancer cell lines does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, and H4ac, a marker of permissive chromatin, we discovered a putative alternative promoter of mir-218-1 in all pancreatic cancer cell lines regardless of KRAS dependence. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity. This suggests that mir-218-1 expression is important and may contain its own tumor suppressor properties independent of SLIT2 signaling. Since pancreatic cancer is an invasive disease we examined the role of miR-218 target genes in specialized cellular structures, termed invadopodia. These structures have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases to degrade extracellular matrix. Transfection of a miR-218 antagomir enhanced invasion in a modified Boyden chamber while addition of mir-218 reduced cell invasion. Our results further demonstrate that mir-218 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that the SLIT2/mir-218-1 signaling axis is an important mediator of pancreatic cancer invasion through the extracellular matrix. Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2014-127

Slit2 is decreased after spontaneous labour in myometrium and regulates pro-labour mediators

Preterm birth, a global healthcare problem, is commonly associated with inflammation. As Slit2 plays an emerging role in inflammation, the purpose of this study was to determine the effect of Slit2 on labour mediators in human gestational tissues. Slit2 mRNA and protein expression were assessed using qRT-PCR and immunohistochemistry in foetal membranes and myometrium obtained before and after labour. Slit2 silencing was achieved using siRNA in primary myometrial cells. Pro-inflammatory and pro-labour mediators were evaluated by qRT-PCR, ELISA and gelatin zymography. Slit2 mRNA and protein expression were found to be significantly lower in myometrium after labour onset. There was no effect of term or preterm labour on Slit2 expression in foetal membranes. Slit2 mRNA expression was decreased in myometrium treated with LPS and IL-1β. Slit2 siRNA in myometrial cells increased IL-1β-induced pro-inflammatory cytokine gene expression and release (IL-6 and IL-8), COX-2 expression and prostaglandin PGE2 and PGF2α release, and MMP-9 gene expression and pro MMP-9 release. There was no effect of Slit2 siRNA on IL-1β-induced NF-κB transcriptional activity. Our results demonstrate that Slit2 is decreased in human myometrium after labour and our knock-down studies describe an anti-inflammatory effect of Slit2 in myometrial cells.

Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury （ALI） and explore the function of Slit2 and Robo4 in ALI. MethodsForty-eight Sprague-Dawley rats were randomly （random number） divided into control group （n = 24） and ALI group （n = 24）. ALI model was reproduced by cecum ligation and puncture （CLP）. The control group only experienced a simulated operation without CLP. Both groups were further divided into 3 subgroups with 8 rats in each subgroup： 12 h, 24 h, and 48 h subgroups, artery blood gas analysis, lung tissue wet/dry weight （W/D） ratio, lung histopathologic changes, pulmonary microvascular permeability were observed. The serum tumor nocrosis factor-α （TNF-α） was measured with enzyme linked immunosorbent assay （ELISA）. The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry. Date were analyzed by one-way ANOVA with SPSS version 13.0 software. Statistical significance was established at a P value of less than 0. 05. Results Compared with the control group, in ALI rats at different time points, partial pressure of oxygen in arterial blood （PaO2） decreased significantly, lung W/D weight ratio and pulmonary microvascular permeability, the serum TNF-α increased significantly （ all P 〈 0. 05 ）, histopathology of lung revealed signs of injury. The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [ （0.56±0.13）vs. （0.87±0.05）, F=41.39, P〈0.05, （0.42±0.10） vs. （0.85±0.07）, F= 93.54, P〈0.05, （0.26±0.08） vs. （0.89±0.09）, F=227.05, P〈0.05]. but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [ （0.86±0.07） vs. （0.83±0.05）, F=0.695, P〉0.05, （0.82±0.05） vs. （0.89±0.08）, F= 2. 061, P 〉 0.05, （0. 86 ± 0.08） vs. （0. 86 ± 0. 05）, F = 0. 035, P 〉 0. 05 ]. Immunohistoehemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells, while lung epithelial cell nuclei and endoehylema. Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells. Compared with the control group, expression of Slit2 protein in lung tissue in ALl group decreased markedly [ （0.37±0.05） vs. （0.45±0.07）, F=6.82, P〈0.05, （0.32±0.06） vs. （0.47±0.09）, F=23.54, P〈0.05, （0.28±0.07） vs. （0.46±0.06）, F= 28.01, P 〈 0. 05 ]. As good as RT-PCR, there were no significant difference in expression of Robo4 protein in lung tissue between two groups [ （0. 53 ±0.04） vs. （0. 52 ±0.05）, F =0. 155, P 〉0. 05, （0. 53 ± 0.09） vs. （0.50±0.05）, F=0.498, P〉0.05, （0.55±0.06） vs. （0.56±0.07）, F=0.073, P 〉 0. 05 ]. Conclusions Lung tissues of control group rats express Slit2 and Robo4. The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALl caused by CLP may be associated with the occurrence of ALl. Key words: Slit2; Robo4; Acute lung injury; Sepsis; Endothelial permeability; Cecum ligation and puncture; TNF-α; Axon guidance cues

Axon Guidance Factor SLIT2 Inhibits Neural Invasion and Metastasis in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting SLIT2-ROBO signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion.

Reduced expression of Slit2 in renal cell carcinoma

Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is down-regulated in RCC partially due to promoter methylation.

SLIT1 Mutation in Patients with Acquired Aplastic Anemia: Its Relevance in Immune Pathophysiology

Abstract 645 Background:Acquired aplastic anemia (AA) is a non-clonal hematopoietic disorder caused by the immune system attack on hematopoietic stem cells (HSCs) and has been thought to be rarely associated with intrinsic HSC defects. However, clonal hematopoiesis by HSCs with genetic alterations may not be uncommon in AA since clonal granulocyte populations showing the paroxysmal nocturnal hemoglobinuria (PNH) phenotype or chromosome 6p uniparental disomy (6pUPD) are detected in approximately 50% and 13% of AA patients, respectively (Katagiri T, et al, Blood, 2011). Abnormal HSCs other than those with the PIG-A mutation or 6pUPD may also contribute to hematopoiesis in AA patients due to immune escape or preferential activation of HSCs with genetic alterations in response to immune pressure in the bone marrow (BM). Objectives/methods:To identify novel genetic abnormalities in patients with AA, we carried out whole exome sequencing of peripheral blood leukocytes from 96 AA patients using a next generation sequencer. Among 40 abnormalities found in more than one patient, we selected abnormalities in genes potentially related to HSC regulation and examined the relevance of the genetic alterations to BM failure. Results:Missense mutations of SLIT1, a family of secreted glycoproteins that bind members of the Robo receptor family and regulate axon guidance, were detected in 15 of 96 patients and confirmed by Sanger sequencing in 3 patients who had been in remission for 1–3 years after immunosuppressive therapy at the time of blood sampling. Since previous studies showed that the Slit-Robo pathway negatively regulates proliferation of murine HSCs as well as a human cancer cell line in an autocrine manner (Prasad A et al., J Biol Chem; 283: 26624. 2008) and the break point of chromosome 10 in a patient with immune-mediated AA possessing t(1:10) was located within the SLIT1 gene, we studied SLIT1 and Robo receptor expression in BM mononuclear cells (BMMCs) obtained from healthy volunteers and patients with AA, and in myeloid leukemia cell lines. Although SLIT1 mRNA was scarce in un-stimulated BMMCs from healthy volunteers, SLIT1 gene expression increased 2.5–3.5 fold when incubated in conditioned medium from PHA or LPS stimulated peripheral blood mononuclear cells. The steady state expression level of SLIT1 by BMMCs from AA patients was 6 fold higher than from healthy individuals. SLIT1 mRNA was also detected in several myeloid leukemia cell lines including K562 and MV4-11, and its expression increased 2 fold by culturing the cells for 48 hours with the human stromal cell line HS-5. Examination of Robo receptor expression using antibodies specific to Robo1-4 showed that BM CD34+ cells from healthy individuals and K562 cells expressed Robo1. Recombinant Slit1 inhibited proliferation of K562 in a dose-dependent manner at concentrations of 1 and 5 mg/ml (Figure 1). Conclusions:The presence of clonal leukocytes with SLIT1 mutations in patients with immune-mediated AA suggests that HSCs with these mutations have survival or proliferation advantages over HSCs without SLIT1 mutations. Slit1 secreted from HSCs or hematopoietic progenitor cells in response to inflammatory cytokines may inhibit their own activation by binding to Robo1, but mutant Slit1 may fail to inhibit activation, leading to preferential commitment of the mutant HSCs to hematopoiesis. The Slit-Robo pathway may therefore serve as a novel therapeutic target for immune-mediated BM failure. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Human Placental Expression of SLIT/ROBO Signaling Cues: Effects of Preeclampsia and Hypoxia1

Preeclampsia is characterized by dysfunctional endothelium and impaired angiogenesis. Recent studies suggest that the neuronal guidance SLIT/ROBO system regulates tumor angiogenesis. This study investigated if SLIT and ROBO are differentially expressed in healthy term and preeclamptic placentas and if hypoxia regulates SLIT and ROBO expression in placental trophoblast and endothelial cells. Total RNA and protein were extracted from placental tissues of healthy term (n = 5) and preeclamptic (n = 6) pregnancies and used for SLIT/ROBO expression analyses with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative-PCR, and immunoblotting. Paraffin-embedded tissues were processed to localize SLIT/ROBO proteins in placental villi by immunohistochemistry. BeWo choriocarcinoma cells and human umbilical vein endothelial cells (HUVEC) were treated with 2% or 10% oxygen or the hypoxia mimetic deferoxamine mesylate (100 μM) to test if hypoxia regulates SLIT/ROBO expression. SLIT2, SLIT3, ROBO1, and ROBO4 mRNA and proteins were detected in the placenta. SLIT2 and ROBO1 proteins localized in the syncytiotrophoblast, and SLIT3, ROBO1, and ROBO4 in capillary endothelium of the placental villi. Levels of ROBO1 and ROBO4 as well as sFLT1 (soluble fms-like tyrosine kinase-1) proteins were significantly greater in preeclamptic placentas compared to normal controls. Hypoxia significantly increased both mRNA and protein levels of SLIT2 in BeWo cells and of SLIT3, ROBO1, and ROBB4 in HUVEC. Thus, trophoblast and endothelial coexpression of SLIT/ROBO suggests an autocrine/paracrine regulatory system for regulating placental function. Differential expression of SLITs and ROBOs in healthy term and preeclamptic placentas and hypoxia regulation of their expressions in placental cells implicate a potential pathophysiological role for this system in preeclampsia.

Developmental alteration of SLIT1 mRNA expression in primate cortex

Developmental alteration of SLIT1 mRNA expression in primate cortex

Prefrontal-Enriched SLIT1 Expression in Old World Monkey Cortex Established during the Postnatal Development

To elucidate the molecular basis of the specialization of cortical architectures, we searched for genes differentially expressed among neocortical areas of Old World monkeys by restriction landmark cDNA scanning . We found that mRNA of SLIT1, an axon guidance molecule, was enriched in the prefrontal cortex but with developmentally related changes. In situ hybridization analysis revealed that SLIT1 mRNA was mainly distributed in the middle layers of most cortical areas, robustly in the prefrontal cortex and faintly in primary sensory areas. The lowest expression was in the primary visual area. Analyses of other SLIT (SLIT2 and SLIT3) mRNAs showed preferential expression in the prefrontal cortex with a distinct laminar pattern. By contrast, the receptor Roundabout (ROBO1 and ROBO2) mRNAs were widely distributed throughout the cortex. Perinatally, SLIT1 mRNA was abundantly expressed in the cortex with modest area specificity. Downregulation of expression initially occurred in early sensory areas around postnatal day 60 and followed in the association areas. The prefrontal area–enriched SLIT1 mRNA expression results from a relatively greater attenuation of this expression in the other areas. These results suggest that its role is altered postnatally and that this is particularly important for prefrontal connectivity in the Old World monkey cortex.