microRNA-365 attenuated intervertebral disc degeneration through modulating nucleus pulposus cell apoptosis and extracellular matrix degradation by targeting EFNA3.

This present study is aimed to investigate the role of microRNA-365 (miR-365) in the development of intervertebral disc degeneration (IDD). Nucleus pulposus (NP) cells were transfected by miR-365 mimic and miR-365 inhibitor, respectively. Concomitantly, the transfection efficiency and the expression level of miRNA were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Meanwhile, NP cells apoptosis was measured through propidium iodide (PI)-AnnexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. Subsequently, immunofluorescence (IF) staining was performed to assess the expression of collagen II, aggrecan and matrix metalloproteinase 13 (MMP-13). In addition, bioinformatic prediction and Luciferase reporter assay were used to reveal the target gene of miR-365. Finally, we isolated the primary NP cells from rats and injected NP-miR-365 in rat IDD models. The results showed that overexpression of miR-365 could effectively inhibit NP cells apoptosis and MMP-13 expression and upregulate the expression of collagen II and aggrecan. Conversely, suppression of miR-365 enhanced NP cell apoptosis and elevated MMP-13 expression, but decreased the expression of collagen II and aggrecan. Moreover, the further data demonstrated that miR-365 mediated NP cell degradation through targeting ephrin-A3 (EFNA3). In addition, the cells apoptosis and catabolic markers were increased in NP cells when EFNA3 upregulated. More importantly, the vivo data supported that miR-365-NP cells injection ameliorated IDD in rats models. miR-365 could alleviate the development of IDD by regulating NP cell apoptosis and ECM degradation, which is likely mediated by targeting EFNA3. Therefore, miR-365 may be a promising therapeutic avenue for treatment IDD through EFNA3.

Ephrin-A3 reverse signaling regulates hippocampal neuronal damage and astrocytic glutamate transport after transient global ischemia.

Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin-A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin-A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin-A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up-regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin-A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus-dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4-mediated ephrin-A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions. Astrocytic ephrin-A3 reverse signaling mediated by EphA4 receptor is necessary for controlling the abundance of glial glutamate transporters under physiological conditions. However, the role of ephrin-A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. We found EphA4-mediated ephrin-A3 reverse signaling to be a crucial mechanism for astrocytes to control glial glutamate transporters and protect hippocampal neurons from glutamate excitotoxicity under ischemic conditions, this cascade representing a potential therapeutic target for stroke.

hMAGEA2 Accelerates the Progression of Prostate Cancer via the EFNA3-Erk1/2 Signaling Pathway.

BACKGROUND/AIM: Human melanoma-associated antigen A2 (hMAGEA2) family members play several roles in many types of cancer and have been explored as potential prognostic markers. In this study, we investigated the molecular mechanism underlying hMAGEA2-mediated tumorigenesis of prostate cancer. MATERIALS AND METHODS: Immunohistochemistry and western blot were used to assess protein expression whereas microarray and quantitative reverse transcription-PCR determined mRNA expression. CCK-8 assay was used to determine cell proliferation. Colony formation assay was used to examine tumorigenesis. Migration and invasion were examined using a transwell assay. Propidium iodide (PI)/Annexin V double staining was performed to measure apoptosis. Transcriptional activity was measured using Dual-luciferase reporter assay. RESULTS: hMAGEA2 was highly over-expressed in human prostate cancer tissues compared to benign prostatic hyperplasia tissues. To elucidate its biological function in prostate cancer, we established two stable hMAGEA2-knockdown prostate cancer cell lines, PC3M and 22RV1, and found that they presented significantly decreased proliferation, anchorage-independent colony formation, migration, and invasion. As hMAGEA2 knockdown suppressed prostate cancer cell growth, we examined its potential influence on tumor apoptosis. hMAGEA2-knockdown cell lines displayed early apoptosis. Moreover, knockdown of hMAGEA2 resulted in the down-regulation of EFNA3 expression. Luciferase assay showed that hMAGEA2 bound to the EFNA promoter region and regulated its transcription. Down-regulation of EFNA3 expression led to decreased Ras/Braf/MEK/Erk1/2 phosphorylation and, consequently, inhibited prostate cancer progression. CONCLUSION: hMAGEA2 promotes prostate cancer growth, metastasis, and tumorigenesis by regulating the EFNA3-Erk1/2 signaling pathway, indicating its potential as a therapeutic marker for prostate cancer.

Clinicopathological and prognostic significance of Ephrin A3 in bladder urothelial carcinoma.

Ephrin A3 (EFNA3) is a member of the Eph/ephrin tyrosine kinase family, which is associated with multiple signaling pathways involved in cell growth and tumor cell metastasis. Aberrant regulation of EFNA3 is associated with the occurrence and development of various types of cancer. However, despite the high incidence of EFNA3 upregulation in cancer, studies concerning EFNA3 in urothelial carcinoma have not, to the best of our knowledge, been conducted. In the present study, bioinformatics analyses using data from multiple online databases were performed to confirm the upregulation of EFNA3 in bladder cancer. The co-expression gene set of EFNA3 and enriched signaling pathways were also analyzed. In addition, immunohistochemistry was conducted to detect EFNA3 expression in 491 clinically confirmed bladder urothelial carcinoma samples and 80 non-cancerous bladder tissues. Kaplan-Meier survival analysis, binary logistic regression analysis, and Cox regression analysis were conducted to confirm the validity of EFNA3 in predicting patient prognosis and its significance in clinical pathology. Statistical analysis demonstrated a significant association between EFNA3 expression levels with tumor size, lymph node metastasis, distant metastasis, and pathological grade. In conclusion, high EFNA3 expression may be a potential biomarker that indicates bladder tumor occurrence and patient prognosis.

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice.

Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2-/- A3-/- mice by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU+ cells in MC derived from A2-/- A3-/- mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho-/-) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2-/- A3-/- , Rho-/- and Rho-/- A2-/- A3-/- mice and the numbers of EdU+ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho-/- mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2-/- A3-/- mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU+ cells in the retinas of adult ephrin-A2-/- A3-/- mice mainly in the inner nuclear layer; and these EdU+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho-/- A2-/- A3-/- mice), a significantly greater amount of EdU+ cells were located in the ciliary body, retina and RPE than that of Rho-/- mice. Comparing between 6 and 12 weeks old Rho-/- A2-/- A3-/- mice, we recorded more EdU+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

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.

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.

Cisplatin chemotherapy-induced miRNA-210 signaling inhibits hepatocellular carcinoma cell growth.

BACKGROUND: Chemotherapy has improved the survival of hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are still not fully understood. MicroRNAs (miRNAs) are critical regulators in carcinogenesis and involved in response to cancer therapy. However, the correlation between miRNAs and chemotherapy is not well-established, and the detailed mechanisms and responsive targets remain unclear. Here, we investigated the function and mechanism of miR-210 in HCC chemotherapy with cisplatin. METHODS: This study involved samples from patients after HCC surgery, including tumor and non-tumor liver tissues. Total RNA was extracted from the fresh tissue samples and the levels of miR-210 were assessed by qRT-PCR analysis. Cisplatin treatment was performed in HepG2 and PLC cell lines, and ephrin A3 (EFNA3) levels were determined by Western blotting. RESULTS: We observed that miR-210 expression was up-regulated in HCC tissues and correlated with HCC progression. Notably, HCC patients underwent recurrences after chemotherapy showed high levels of miR-210 expression in tumors, indicating that miR-210 might be involved in regulating the chemotherapeutic efficacy. We also demonstrated that cisplatin treatment decreased the expression of miR-210 and increased the expression of miR-210 target EFNA3 in HCC cells. Moreover, miR-210 overexpression prevented the effects of cisplatin and rescued HCC cell growth, and miR-210 inhibition contributed to improved chemosensitivity of cisplatin in HCC cells. CONCLUSIONS: Our findings defined a novel mechanism underlying the efficacious effects of cisplatin chemotherapy in HCC, and miR-210-induced EFNA3 signaling might be a potential target of cisplatin in HCC treatment.

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.

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.