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.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Small extracellular vesicles derived from hypoxic mesenchymal stem cells promote vascularized bone regeneration through the miR-210-3p/EFNA3/PI3K pathway.

Angiogenesis is closely coupled with osteogenesis and has equal importance. Thus, promoting angiogenesis during the bone repair process is vital for ideal bone regeneration. As important mediators of cell-cell communication and biological homeostasis, mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proved to be highly involved in bone and vascular regeneration. Because hypoxia microenvironment promotes the proangiogenic activity of MSCs, in the present study, we investigate the effect and underlying molecular mechanisms of sEVs from hypoxia-preconditioned MSCs (hypo-sEVs) on angiogenesis and develop an effective strategy to promote vascularized bone regeneration. Compared to sEVs from normoxia MSCs (nor-sEVs), hypo-sEVs promoted the proliferation, migration, and angiogenesis of HUVECs and ultimately enhanced bone regeneration and new blood vessel reconstruction in a critical-size calvarial bone defect model. miRNA sequence and the verified results showed that miR-210-3p in hypo-sEVs was increased via HIF-1α under hypoxia. The upregulated miR-210-3p in hypo-sEVs promoted angiogenesis by downregulating EFNA3 expression and enhancing the phosphorylation of the PI3K/AKT pathway. Thus, this study suggests a successful strategy to enhance vascularized bone regeneration by utilizing hypo-sEVs via the miR-210-3p/EFNA3/PI3K/AKT pathway. STATEMENT OF SIGNIFICANCE: Considering the significance of vascularization in ideal bone regeneration, strategies to promote angiogenesis during bone repair are required. Hypoxia microenvironment can promote the proangiogenic potential of mesenchymal stem cells (MSCs). Nonetheless, the therapeutic effect of small extracellular vesicles (sEVs) from hypoxia-preconditioned MSCs on cranio-maxillofacial bone defect remains unknown, and the underlying mechanism is poorly understood. This study shows that hypo-sEVs significantly enhance the proliferation, migration, and angiogenesis of HUVECs as well as promote vascularized bone formation. Moreover, this work indicates that HIF-1α can induce overexpression of miR-210-3p under hypoxia, and miR-210-3p can hinder EFNA3 expression and subsequently activate the PI3K/AKT pathway. The application of hypo-sEVs provides a facile and promising strategy to promote vascularized bone regeneration in a critical-size bone defect model.

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.

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.

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.

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.

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.

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.

Ephrin-A3 and ephrin-A4 contribute to microglia-induced angiogenesis in brain endothelial cells.

The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia-deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary-like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin-A3 and ephrin-A4, were specifically upregulated in BMECs exposed to BV2-derived culture supernatant. Knockdown of ephrin-A3 and ephrin-A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF-α) released from BV2 microglial cells. Moreover, the upregulations of ephrin-A3 and ephrin-A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF-α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin-A3 and ephrin-A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia-released TNF-α.

WSS25 inhibits Dicer, downregulating microRNA-210, which targets Ephrin-A3, to suppress human microvascular endothelial cell (HMEC-1) tube formation.

WSS25 is a sulfated polysaccharide that inhibits angiogenesis. However, the mechanism underlying the regulation of angiogenesis by WSS25 is not well understood. Using microRNA (miRNA) microarray analysis, a total of 25 miRNAs were found to be upregulated and 12 (including miR-210) downregulated by WSS25 in human microvascular endothelial cells (HMEC-1). Interestingly, Dicer, a key enzyme for miRNA biosynthesis, was downregulated by WSS25 in HMEC-1 cells. Further studies indicated that HMEC-1 cell tube formation and miR-210 expression were suppressed while Ephrin-A3 expression was enhanced by the silencing of Dicer. In contrast, HMEC-1 cell tube formation and miR-210 expression were induced while Ephrin-A3 expression was suppressed by Dicer overexpression. Moreover, miR-210 was downregulated while Ephrin-A3 was upregulated by WSS25 in HMEC-1 cells. HMEC-1 cell migration and tube formation were arrested, while Ephrin-A3 expression was augmented by anti-miR-210. In addition, HMEC-1 cell tube formation was significantly attenuated or augmented when Ephrin-A3 was overexpressed or silenced, respectively. Nevertheless, the tube formation blocked by WSS25 could be partially rescued by manipulation of Dicer, miR-210 and Ephrin-A3. These results suggest a new pathway whereby WSS25 inhibits angiogenesis via suppression of Dicer, leading to downregulation of miR-210 and upregulation of Ephrin-A3.

MicroRNA-210 promotes proliferation and invasion of peripheral nerve sheath tumor cells targeting EFNA3.

MicroRNA (miR) plays an important role in tumorigenesis including malignant peripheral nerve sheath tumor (MPNST). miR-210 downregulation is frequently observed in a variety of tumors. In this study, miR-210 was identified as downregulated in MPNST cells, and its potential target ephrin-A3 (EFNA3) was upregulated in them compared with neurofibroma cells using quantitative real-time (qRT)-PCR. Luciferase reporter assay further demonstrates that EFNA3 is a target of miR-210. Then it is confirmed that miR-210 can regulate EFNA3 mRNA and protein expression in MPNST ST88-14 (NF1 wild-type) and sNF96.2 (NF1 mutant type) cell lines. The functions of miR-210 in MPNST cells were investigated, and the results showed that overexpression of miR-210 increased cellular viability, colony formation, S phase percentage, and invasiveness of MPNST cells. Inversely, inhibition of miR-210 expression induced suppression of proliferation and invasion of MPNST cells. These results suggest that miR-210-mediated EFNA3 promotion of proliferation and invasion of MPNST cells plays an important role in MPNST tumorigenesis and progression. miR-210 and EFNA3 may be candidate novel therapeutic targets for MPNST.