Long-term impact of maternal obesity on the gliovascular unit and ephrin signaling in the hippocampus of adult offspring.

BACKGROUND: Children born to obese mothers are at increased risk of developing mood disorders and cognitive impairment. Experimental studies have reported structural changes in the brain such as the gliovascular unit as well as activation of neuroinflammatory cells as a part of neuroinflammation processing in aged offspring of obese mothers. However, the molecular mechanisms linking maternal obesity to poor neurodevelopmental outcomes are not well established. The ephrin system plays a major role in a variety of cellular processes including cell-cell interaction, synaptic plasticity, and long-term potentiation. Therefore, in this study we determined the impact of maternal obesity in pregnancy on cortical, hippocampal development, vasculature and ephrin-A3/EphA4-signaling, in the adult offspring in mice. METHODS: Maternal obesity was induced in mice by a high fat/high sugar Western type of diet (HF/HS). We collected brain tissue (prefrontal cortex and hippocampus) from 6-month-old offspring of obese and lean (control) dams. Hippocampal volume, cortical thickness, myelination of white matter, density of astrocytes and microglia in relation to their activity were analyzed using 3-D stereological quantification. mRNA expression of ephrin-A3, EphA4 and synaptic markers were measured by qPCR in the brain tissue. Moreover, expression of gap junction protein connexin-43, lipocalin-2, and vascular CD31/Aquaporin 4 were determined in the hippocampus by immunohistochemistry. RESULTS: Volume of hippocampus and cortical thickness were significantly smaller, and myelination impaired, while mRNA levels of hippocampal EphA4 and post-synaptic density (PSD) 95 were significantly lower in the hippocampus in the offspring of obese dams as compared to offspring of controls. Further analysis of the hippocampal gliovascular unit indicated higher coverage of capillaries by astrocytic end-feet, expression of connexin-43 and lipocalin-2 in endothelial cells in the offspring of obese dams. In addition, offspring of obese dams demonstrated activation of microglia together with higher density of cells, while astrocyte cell density was lower. CONCLUSION: Maternal obesity affects brain size, impairs myelination, disrupts the hippocampal gliovascular unit and decreases the mRNA expression of EphA4 and PSD-95 in the hippocampus of adult offspring. These results indicate that the vasculature-glia cross-talk may be an important mediator of altered synaptic plasticity, which could be a link between maternal obesity and neurodevelopmental/neuropsychiatric disorders in the offspring.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

BACKGROUND & AIMS: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. METHODS: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. RESULTS: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. CONCLUSIONS: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

LncRNA LINC01270 aggravates the progression of gastric cancer through modulation of miR-326/EFNA3 axis.

Gastric cancer (GC) is lethal malignancy, which is associated with high mortality. Long noncoding RNA LINC01270 has been identified to act as a potential oncogene in several cancers. However, its role and related regulatory mechanism in GC are yet to be illustrated. The levels of lncRNA LINC01270, miR-326, and EphrinA3 (EFNA3) were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) and colony formation assays were applied for analyzing cell proliferation. Transwell assay was used for measuring cellular migration and invasion. Western blot analysis was employed for evaluating the protein levels. Luciferase reporter and RNA pull-down assays were utilized to verify the binding ability between LINC01270 (or EFNA3) and miR-326. Our findings indicated that LINC01270 expression was significantly up-regulated in GC tissues and cell lines. Additionally, LINC01270 knockdown attenuated GC progression through inhibiting cell proliferation, migration, and invasion. Functional experiments identified that lncRNA LINC01270 could positively regulate EFNA3 expression by serving as a competing endogenous RNA (ceRNA) for miR-326. Through rescue assays, inhibition of GC progression caused by LINC01270 suppression was found to be reversed by the application of miR-326 inhibitor or EFNA3 overexpression. Overall, our work demonstrated that lncRNA LINC01270 can accelerate cell proliferation, migration, and invasion via modulating miR-326/EFNA3 axis. These findings might implicate the potential role of lncRNA LINC01270 in GC treatment.

Ephrin-A3 is required for tonotopic map precision and auditory functions in the mouse auditory brainstem.

Tonotopy is a prominent feature of the vertebrate auditory system and forms the basis for sound discrimination, but the molecular mechanism that underlies its formation remains largely elusive. Ephrin/Eph signaling is known to play important roles in axon guidance during topographic mapping in other sensory systems, so we investigated its possible role in the establishment of tonotopy in the mouse cochlear nucleus. We found that ephrin-A3 molecules are differentially expressed along the tonotopic axis in the cochlear nucleus during innervation. Ephrin-A3 forward signaling is sufficient to repel auditory nerve fibers in a developmental stage-dependent manner. In mice lacking ephrin-A3, the tonotopic map is degraded and isofrequency bands of neuronal activation upon pure tone exposure become imprecise in the anteroventral cochlear nucleus. Ephrin-A3 mutant mice also exhibit a delayed second wave in auditory brainstem responses upon sound stimuli and impaired detection of sound frequency changes. Our findings establish an essential role for ephrin-A3 in forming precise tonotopy in the auditory brainstem to ensure accurate sound discrimination.

OSCC Exosomes Regulate miR-210-3p Targeting EFNA3 to Promote Oral Cancer Angiogenesis through the PI3K/AKT Pathway.

This study is aimed at determining how oral squamous cell carcinoma (OSCC) regulates the angiogenesis of HUVECs through miR-210-3p expression and exploring the relationship among miR-210-3p, its target protein, and the possible mechanism of angiogenesis regulation. miR-210-3p expression was detected in OSCC tissues and juxta cancerous tissues (JCT), and the relationship among miR-210-3p, microvessel density (MVD), and histopathologic features was analyzed. A conditioned medium (CM) of the OSCC cell line CAL27 was collected to stimulate human umbilical vein endothelial cells (HUVECs), and the miR-210-3p levels and tube formation capability of HUVECs were measured. The target protein level of miR-210-3p was altered; then, PI3K/AKT pathway activation in HUVECs was detected. miR-210-3p was tested in exosomes separated from CAL27 CM, and the transfer of miR-210-3p from OSCC exosomes to HUVECs was verified. Then, we found that the OSCC tissues had higher miR-210-3p levels than the JCT, and miR-210-3p level was positively correlated with MVD and tumor grade. CAL27 CM was able to elevate miR-210-3p levels in HUVECs and promoted tube formation. EFNA3 was the target gene of miR-210-3p, and ephrinA3 protein level was able to influence the migration and proliferation of HUVECs. The levels of phosphorylated AKT in the HUVECs increased when ephrinA3 was downregulated, and the upregulation of ephrinA3 resulted in the suppression of the PI3K/AKT pathway. miR-210-3p was detected in exosomes isolated from the CM of CAL27 cells, and miR-210-3p level in the HUVECs was elevated after absorbing the OSCC exosomes. In conclusion, miR-210-3p was more overexpressed in OSCC tissues than in the JCT. The exosomes secreted by OSCC cells were able to upregulate miR-210-3p expression and reduce ephrinA3 expression in HUVECs and promoted tube formation through the PI3K/AKT signaling pathway.

Fluoxetine and Riluzole Mitigates Manganese-Induced Disruption of Glutamate Transporters and Excitotoxicity via Ephrin-A3/GLAST-GLT-1/Glu Signaling Pathway in Striatum of Mice.

Manganese (Mn) is an essential element required for many biological processes and systems in the human body. Mn intoxication increases brain glutamate (Glu) levels causing neuronal damage. Recent studies have reported that ephrin-A3 regulates this glutamate transporter. However, none has explored the role of this crucial molecule in Mn-induced excitotoxicity. The present study investigated whether ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity using astrocytes and Kunming mice. The mechanisms were explored using fluoxetine (ephrin-A3 inhibitor) and riluzole (a Glu release inhibitor). Firstly, we demonstrated that Mn exposure (500 µM or 50 mg/kg MnCl2) significantly increased Mn, ephrin-A3, and Glu levels, and inhibited Na+-K+ ATPase activity, as well as mRNA and protein levels of GLAST and GLT-1. Secondly, we found that astrocytes and mice pretreated with fluoxetine (100 µM or 15 mg/kg) and riluzole (100 µM or 32 µmol/kg) prior to Mn exposure had lower ephrin-A3 and Glu levels, but higher Na+-K+ ATPase activity, expression levels of GLAST and GLT-1 than those exposed to 500 µM or 50 mg/kg MnCl2. Moreover, the morphology of cells and the histomorphology of mice striatum were injured. Results showed that pretreatment with fluoxetine and riluzole attenuated the Mn-induced motor dysfunctions. Together, these results suggest that the ephrin-A3/GLAST-GLT-1/Glu signaling pathway participates in Mn-induced excitotoxicity, and fluoxetine and riluzole can mitigate the Mn-induced excitotoxicity in mice brain.

MiR-210-3p-EphrinA3-PI3K/AKT axis regulates the progression of oral cancer.

This study aimed to explore new therapeutic targets to improve the survival rate of patients with oral squamous cell carcinoma (OSCC).MiR-210-3p, EphrinA3 and EMT related indices were evaluated in OSCC tissues and cell lines. In addition, the relationship between differential EphrinA3 expression and tumour progression was explored through molecular biology techniques, in vitro functional experiments and tumour xenotransplantation models. The expression of EphrinA3 (rs  = -0.719, P < .05) and E-cadherin (rs  = -0.856, P < .05) was negatively correlated with the pathological grading in OSCC tissues. Protein clustering shows EphrinA3 may be associated with tumour progression. EphrinA3 also can regulate the biological behaviour of oral cancer cells. And it regulates the EMT by the PI3K/AKT signalling pathway. MiR-210-3p targeted the gen EFNA3. Up-regulation of miR-210-3p expression can decrease the expression of EphrinA3 and further to influence the biological behaviour of OSCC. The miR-210-3p-EphrinA3-PI3K/AKT signalling axis plays an important role in the progress of OSCC. EphrinA3 may serve as a novel target for oral cancer treatment.

Optimization of miRNA delivery by using a polymeric conjugate based on deoxycholic acid-modified polyethylenimine.

Safe and efficient delivery of microRNA (miRNA) molecules is essential for their successful transition from research to the clinic setting. In the present study, we have used a bile acid, deoxycholic acid (DA), to modify 1.8 kDa branched polyethylenimine (bPEI1.8) and subsequently investigated gene delivery features of the resultant conjugates (PEI-DAn). We found significant differences between the PEI-DAn conjugates and conventional bPEIs with respect to miRNA condensation ability, buffering capacity, cellular uptake, and intracellular gene release behavior in endothelial cells (ECs) isolated from human umbilical vein (HUVECs). Changes in the conjugation degree greatly influenced the transfection performance of the PEI-DAn conjugates with respect to miRNA condensation and decondensation properties as well as cellular uptake behavior. The PEI-DA3 conjugates could significantly enhance the expression level of miRNA-210 in HUVECs. The overexpressed miRNA-210, in turn, markedly downregulated the expression levels of Efna3 and Ptp1b as well as led to a substantial rise in HUVECs' migration rate in a wound healing assay. Collectively, our results have demonstrated that PEI-DA3 conjugates facilitate the formation of stable nanocomplexes that are loose enough to release miRNAs into the cytosol. The free bioavailable miRNAs, in turn, result in efficient gene silencing comparable to bPEI25 as well as Lipofectamine RNAiMAX.

Deletion of long noncoding RNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulation of miR-101a.

Long noncoding RNAs (lncRNAs) have been reported to be involved in several neurological pathogenesis conditions including cerebral ischemia. In the current study, the functions of lncRNA EFNA3 on hypoxia-injured rat adrenal pheochromocytoma (PC-12) cells and the underlying molecular mechanism were studied. The expression of lncRNA EFNA3 was silenced by short hairpin RNA transfection, after which the cells were subjected with hypoxia. Cell viability, migration, invasion, and apoptosis were, respectively, determined by trypan blue staining, Transwell assay, annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining, and Western blot analysis. The cross regulation between lncRNA EFNA3 and miR-101a, as well as between miR-101a and Rho associated coiled-coil containing protein kinase 2 (ROCK2) were detected by performing quantitative real-time polymerase chain reaction, RNA pull-down, RNA immunoprecipitation, luciferase activity assay, and Western blot analysis. Studies showed that lncRNA EFNA3 was highly expressed in response to hypoxia. Deletion of lncRNA EFNA3 significantly aggravated hypoxia-induced injury in PC-12 cells, as the impairment of cell viability, migration, and invasion, and the inducement of apoptosis. LncRNA EFNA3 worked as a sponging molecule for miR-101a and miR-101a suppression-protected PC-12 cells against hypoxia-induced injury even when lncRNA EFNA3 was silenced. ROCK2 was a target gene of miR-101a. ROCK2 overexpression exhibited neuroprotective activities. Besides, ROCK2 overexpression activated Wnt/ß-catenin pathway whereas it deactivated JAK/STAT pathway upon hypoxia. Our study suggests that deletion of lncRNA EFNA3 aggravates hypoxia-induced injury in PC-12 cells by upregulating miR-101a, which further targets ROCK2.

Loss of the Spinocerebellar Ataxia type 3 disease protein ATXN3 alters transcription of multiple signal transduction pathways.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disorder caused by a polyglutamine-encoding CAG repeat expansion in the ATXN3 gene which encodes the deubiquitinating enzyme, ATXN3. Several mechanisms have been proposed to explain the pathogenic role of mutant, polyQ-expanded ATXN3 in SCA3 including disease protein aggregation, impairment of ubiquitin-proteasomal degradation and transcriptional dysregulation. A better understanding of the normal functions of this protein may shed light on SCA3 disease pathogenesis. To assess the potential normal role of ATXN3 in regulating gene expression, we compared transcriptional profiles in WT versus Atxn3 null mouse embryonic fibroblasts. Differentially expressed genes in the absence of ATXN3 contribute to multiple signal transduction pathways, suggesting a status switch of signaling pathways including depressed Wnt and BMP4 pathways and elevated growth factor pathways such as Prolactin, TGF-ß, and Ephrin pathways. The Eph receptor A3 (Efna3), a receptor protein-tyrosine kinase in the Ephrin pathway that is highly expressed in the nervous system, was the most differentially upregulated gene in Atxn3 null MEFs. This increased expression of Efna3 was recapitulated in Atxn3 knockout mouse brainstem, a selectively vulnerable brain region in SCA3. Overexpression of normal or expanded ATXN3 was sufficient to repress Efna3 expression, supporting a role for ATXN3 in regulating Ephrin signaling. We further show that, in the absence of ATXN3, Efna3 upregulation is associated with hyperacetylation of histones H3 and H4 at the Efna3 promoter, which in turn is induced by decreased levels of HDAC3 and NCoR in ATXN3 null cells. Together, these results reveal a normal role for ATXN3 in transcriptional regulation of multiple signaling pathways of potential relevance to disease processes in SCA3.

Regulatory roles of ephrinA5 and its novel signaling pathway in mouse primary granulosa cell apoptosis and proliferation.

Recent findings suggest that ephrinA5 (Efna5) has a novel role in female mouse fertility, in addition to its well-defined role as a neurogenesis factor. Nevertheless, its physiological roles in ovarian granulosa cells (GC) have not been determined. In this study, mouse GC were cultured and transfected with ephrin A5 siRNA and negative control to determine the effects of Efna5 on GC apoptosis, proliferation, cell cycle progression, and related signaling pathways. To understand the mode signaling, the mRNA expression levels of Efna5 receptors (Eph receptor A5, Eph receptor A3, Eph receptor A8, and Eph receptor B2) were examined. Both mRNA and protein expressions of apoptosis-related factors (Bax, Bcl-2, Caspase 8, Caspase 3, and Tnfα) and a proliferation marker, Pcna, were investigated. Additionally, the role of Efna5 on paracrine oocyte-secreted factors and steroidogenesis hormones were also explored. Efna5 silencing suppressed GC apoptosis by downregulating Bax and upregulating Bcl-2 in a Caspase 8-dependent manner. Efna5 knockdown promoted GC proliferation via p-Akt and p-ERK pathway activation. The inhibition of Efna5 enhanced BMH15 and estradiol expression, but suppressed GDF9, while progesterone level remained unaltered. These results demonstrated that Efna5 is a pro-apoptotic agent in GC and plays important role in folliculogenesis by mediating apoptosis, proliferation, and steroidogenesis in female mouse. Therefore Efna5 might be potential therapeutic target for female fertility disorders.

The role of miR-210, E2F3 and ephrin A3 in angiosarcoma cell proliferation.

BACKGROUND: Although angiosarcoma exhibits aggressive progression and is associated with unfavourable prognosis, its pathogenesis is poorly understood. OBJECTIVES: In the present study, we investigated the possibility that microRNAs play a role in the pathogenesis of angiosarcoma. MATERIALS & METHODS: microRNA expression was evaluated by array analysis and real-time PCR, and protein expression was determined by immunohistochemistry and immunoblotting. RESULTS: miR-210 expression was decreased in angiosarcoma cells both in vivo and in vitro. E2F3 and ephrin A3 are putative targets of miR-210, and their protein expression was up-regulated in the tumour cells. Knockdown of E2F3 or ephrin A3 resulted in a significant decrease in the number of angiosarcoma cells. CONCLUSION: Further investigations into the regulatory mechanisms of oncogenesis associated with miR-210/E2F3/ephrin A3 signalling may lead to a new therapeutic approach against angiosarcoma.

Hypoxia-induced miR-210 promoter demethylation enhances proliferation, autophagy and angiogenesis of schwannoma cells.

Hypoxia, a dominant feature in cancer occurrence and evolution, exists throughout the progression of most malignant tumors. This study focused on the mechanism of hypoxia-induced miR-210 upregulation, and the miR-210 functions in schwannoma. We detected microvascular density, vascular endothelial growth factor (VEGF) and miR-210 expression levels using schwannoma tissue mciroarray. The results showed that miR-210 expression was significantly associated with VEGF. Moreover, the cytological tests showed that hypoxia induced miR-210 expression, while reduce ephrin-A3 expression. The bisulfate genomic sequencing PCR results showed that miR-210 promoter region was hypermethylated in RT4-D6P2T in normoxia, while demethylated in hypoxia, and the region included the hypoxia-inducible factor-1α (HIF-1α) response element site. Cellular function research showed that hypoxia resulted in RT4-D6P2T apoptosis, higher autophage and invasion. Besides, hypoxia can affect HIF-1α/VEGF-mediated angiogenesis. To learn about the specific functions of miR-210, we found that with miR-210 inhibition, tumor cell apoptosis increased, autophagy and angiogenesis reduced, and the cell cycle was arrested. Hypoxia promoted miR-210 expression through promoter demethylation, then consequently enhanced tumor cell proliferation and autophagy, increasing tumor cell angiogenesis. Thus, miR-210 could be a potential marker for judging tumor malignancy and be taken as an effective target for clinical auxiliary treatment of neurilemmoma.

Visualization of exosome-mediated miR-210 transfer from hypoxic tumor cells.

Cancer cells actively release exosomes carrying specific cellular components, such as proteins, mRNA, and miRNA, to communicate with various cells in the tumor microenvironment. We visualized exosome-mediated transfer of miR-210 from hypoxic breast cancer cells to neighboring cells using a miR-210 specific reporter system. By in vitro and in vivo visualization, we found that exosomes with miR-210 were transferred to cells in the tumor microenvironment and that miR-210 was involved in expression of vascular remodeling related genes, such as Ephrin A3 and PTP1B, to promote angiogenesis. These results indicate that cellular components, such as miRNAs from hypoxic cancer cells, spread to adjacent cancer cells in the tumor microenvironment via exosomes and influence tumor progression.

Axon guidance molecule expression after cell therapy in a mouse model of Parkinson's disease.

BACKGROUND: Cell therapy is a promising approach for Parkinson's disease (PD). Others and we have previously shown that transplantation of ventral mesencephalic fetal cells into substantia nigra (SN) in an animal model of PD enables anatomical and functional repair of the degenerated pathway. However, the molecular basis of this repair is still largely unknown. OBJECTIVE: In this work, we studied the expression of several axon guidance molecules that may be implicated in the repair of the degenerated nigrostriatal pathway. METHODS: The expression of axon guidance molecules was analyzed using qRT-PCR on five specific regions surrounding the nigrostriatal pathway (ventral mesencephalon (VM), thalamus (Thal), medial forebrain bundle (MFB), nucleus accumbens (NAcc) and caudate putamen (CPu)), one and seven days after lesion and transplantation. RESULTS: We showed that mRNA expression of specific axon guidance molecules and their receptors is modified in structures surrounding the nigrostriatal pathway, suggesting their involvement in the axon guidance of grafted neurons. Moreover, we highlight a possible new role for semaphorin 7A in this repair. CONCLUSION: Overall, our data provide a reliable basis to understand how axons of grafted neurons are able to navigate towards their targets and interact with the molecular environment in the adult brain. This should help to improve the efficiency of cell replacement approaches in PD.

Inverse Expression Levels of EphrinA3 and EphrinA5 Contribute to Dopaminergic Differentiation of Human SH-SY5Y Cells.

Two key principles underlying successful cellular therapies for Parkinson's disease (PD) are appropriate differentiation of dopaminergic (DA) neurons from transplanted cells and precise axon growth. EphrinAs, a subclass of ephrins, act as axon guidance molecules and are highly expressed in DA brain regions. Existing evidences indicate that they act as either repulsion or attraction signals to guide axon growth. This study investigated whether ephrinAs are involved in DA neuron differentiation. Data from miRCURY™ LNA mRNAs/microRNAs microarrays and quantitative real-time polymerase chain reaction (qRT-PCR) showed upregulated ephrinA3 mRNA (EFNA3) and downregulated ephrinA5 mRNA (EFNA5) during DA neuron differentiation. In addition, hsa-miR-4271 was downregulated, which could influence EFNA3 translation. Furthermore, immunofluorescence (IF) and western blotting confirmed the mRNA results and showed increased ephrinA3 and decreased ephrinA5 protein levels in differentiating DA neurons. Taken together, our results indicate that inverse expression levels of ephrinA3 and ephrinA5, which are possibly influenced by microRNAs, contribute to DA neuron differentiation by guiding axon growth.

EGCG ameliorates the hypoxia-induced apoptosis and osteogenic differentiation reduction of mesenchymal stem cells via upregulating miR-210.

The healing process of fractured bone is affected by the multiple factors regulating the growth and differentiation of osteoblasts and bone mesenchymal stem cells (MSCs), however, such markers and molecular events need to be orchestrated in details. This study investigated the effect of polyphenol(-)-epigallocatechin-3-gallate (EGCG) on the hypoxia-induced apoptosis and osteogenic differentiation of human bone marrow-derived MSCs, examined the miR-210 induction by EGCG, explored the target inhibition of the expression of receptor tyrosine kinase ligand ephrin-A3 (EFNA3) by miR-210, and then determined the association of the miR-210 promotion with the hypoxia-induced apoptosis and osteogenic differentiation. Results demonstrated that EGCG treatment significantly inhibited the hypoxia-induced apoptosis in MSCs and promoted the level of alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), propeptide of type I procollagen I (PINP) and runt-related transcription factor 2 (RUNX2) in MSCs under either normoxia or hypoxia. Moreover, the EGCG treatment upregulated the miR-210 expression, in an association with EFNA3 downregulation; and the miR-210 upregulation significantly downregulated the expression of EFNA3 via the specific binding to the 3' UTR of EFNA3. In addition, the manipulated miR-210 upregulation exerted amelioration on the hypoxia-induced apoptosis and on the hypoxia-reduced expression of ALP, BMP-2, PINP and RUNX2 in MSCs. In summary, our study indicated the protective role of EGCG in response to hypoxia and promontory role to osteogenic differentiation in MSCs via upregulating miR-210 and downregulating the expression of miR-210-targeted EFNA3. Our study implies the protective role of EGCG in the hypoxia-induced impairment in MSCs.

Dissecting the roles of Ephrin-A3 in malignant peripheral nerve sheath tumor by TALENs.

Malignant peripheral nerve sheath tumor (MPNST) is a rare and aggressive soft tissue sarcoma for which effective treatments have not yet been established due to poor understanding of its pathogenesis. Our previous study indicated that miR-210-mediated Ephrin-A3 (EFNA3) promotion of proliferation and invasion of MPNST cells plays an important role in MPNST tumorigenesis and progression. The purpose of the present study was to further investigate the roles of EFNA3 in MPNST. Constructed transcription activator-like effector nucleases (TALENs) and lentiviral vectors were transfected into MPNST ST88-14 (NF1 wild-type) and sNF96.2 (NF1 mutant type) cell lines to obtain gain- and loss-of-function cell lines for the EFNA3 function study. The results showed that the knockout of ENFA3 increased cellular viability and invasiveness of the MPNST cells. However, the adhesion ability of MPNST cells was enhanced or inhibited when EFNA3 was overexpressed or knocked out, respectively. It was also observed that knockout of EFNA3 significantly decreased the expression of phosphorylated FAK (p-FAK) and the tumor necrosis factor α (TNF-α) compared to that in the control cells, yet the expression of phosphatidylinositol 3-kinase (PI3K), GTPase, integrins, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-α) increased significantly. Inversely, overexpression of EFNA3 significantly increased the expression of p-FAK and TNF-α compared to that in the control cells, yet the expression of PI3K, GTPase, integrins, VEGF and HIF-α decreased significantly. The results indicated that EFNA3 serves as a tumor suppressor in MPNST cells and it may play a critical role in the focal adhesion kinase (FAK) signaling and VEGF-associated tumor angiogenesis pathway. These findings may not only facilitate the better understanding of MPNST pathogenesis, but also suggest EFNA3 as a promising target for MPNST treatment.

Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders.

EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms.

EFNA3 long noncoding RNAs induced by hypoxia promote metastatic dissemination.

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.