RNA Modification-Related Genetic Variants in Genomic Loci Associated with Bone Mineral Density and Fracture.

Genome-wide association studies (GWASs) have identified more than 500 loci for bone mineral density (BMD), but functional variants in these loci are less known. The aim of this study was to identify RNA modification-related SNPs (RNAm-SNPs) for BMD in GWAS loci. We evaluated the association of RNAm-SNPs with quantitative heel ultrasound BMD (eBMD) in 426,824 individuals, femoral neck (FN) and lumbar spine (LS) BMD in 32,961 individuals and fracture in ~1.2 million individuals. Furthermore, we performed functional enrichment, QTL and Mendelian randomization analyses to support the functionality of the identified RNAm-SNPs. We found 300 RNAm-SNPs significantly associated with BMD, including 249 m6A-, 28 m1A-, 3 m5C-, 7 m7G- and 13 A-to-I-related SNPs. m6A-SNPs in OP susceptibility genes, such as WNT4, WLS, SPTBN1, SEM1, FUBP3, LRP5 and JAG1, were identified and functional enrichment for m6A-SNPs in the eBMD GWAS dataset was detected. eQTL signals were found for nearly half of the identified RNAm-SNPs, and the affected gene expression was associated with BMD and fracture. The RNAm-SNPs were also associated with the plasma levels of proteins in cytokine-cytokine receptor interaction, PI3K-Akt signaling, NF-kappa B signaling and MAPK signaling pathways. Moreover, the plasma levels of proteins (CCL19, COL1A1, CTSB, EFNA5, IL19, INSR, KDR, LIFR, MET and PLXNB2) in these pathways were found to be associated with eBMD in Mendelian randomization analysis. This study identified functional variants and potential causal genes for BMD and fracture in GWAS loci and suggested that RNA modification may play an important role in osteoporosis.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Identification and Validation of a Dysregulated miRNA-Associated mRNA Network in Temporal Lobe Epilepsy.

OBJECTIVES: This study is aimed at exploring the relationships between miRNAs and mRNAs and to characterize their biological functions in temporal lobe epilepsy (TLE). METHODS: Novel clinical significant miRNAs and target genes and their potential underlying mechanisms have been discovered and explored by mining miRNAs and mRNA expression data of TLE patients using various bioinformatics methods. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to validate the bioinformatic analysis results. RESULTS: A total of 6 dysregulated miRNAs and 442 differentially expressed genes (DEGs) related to TLE were obtained from GEO database (GSE114701 and GSE127871 datasets). A protein-protein interaction (PPI) network containing the 442 DEGs was established. mRNA response elements from the 6 dysregulated miRNAs were predicted using the miRDB and TargetScan bioinformatic tools. By merging the identified targets of the dysregulated miRNAs and the 247 downregulated DEGs, a miRNA-mRNA network was constructed revealing the interaction of miR-484 with eight mRNAs (ABLIM2, CEP170B, CTD-3193O13.9, EFNA5, GAP43, PRKCB, FXYD7, and NCAN). A weighted correlation network analysis (WGCNA) based on the eight genes was established and demonstrated that these mRNAs, except FXYD7 and NCAN, were hub genes in the network. Gene Oncology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the six hub genes were mainly involved in cellular-related biological functions and the neurotransmitter synapse pathway. The differences in expression levels of the miR-484 and the three hub genes (CTD-3193O13.9, EFNA5, and PRKCB) observed experimentally in TLE patients compared to those of healthy controls were consistent with the WGCNA prediction. CONCLUSION: Our study suggests that understanding the miRNA-mRNA interactions will provide insights into the epilepsy pathogenesis. In addition, our results indicate that miR-484 may be a promising novel biomarker for TLE.

PIWIL1 interacting RNA piR-017061 inhibits pancreatic cancer growth via regulating EFNA5.

PIWI (P element induced wimpy testis) integrating RNAs (piRNAs) are small non-coding RNAs with the length of approximately 30 nucleotides that plays crucial roles in germ cells and adult stem cells. Recently, accumulating data have shown that piRNA and PIWI proteins are involved in tumorigenesis. However, the roles of PIWI proteins and piRNAs in pancreatic cancer are still elusive. Here, we showed that piR-017061 is significantly downregulated in pancreatic cancer patients' samples and pancreatic cancer cell lines. Furthermore, we studied the function of piR-017061 in pancreatic cancer and our data revealed that piR-017061 inhibits pancreatic cancer cell growth in vitro and in vivo. Moreover, we analyzed the genomic loci around piR-017061 and identified EFNA5 as a novel target of piR-017061. Importantly, our data further revealed a direct binding between piR-017061 and EFNA5 mRNA mediated by PIWIL1. Mechanically, piR-017061 cooperates with PIWIL1 to facilitate EFNA5 mRNA degradation and loss of piR-017061 results in accumulation of EFNA5 which facilitates pancreatic cancer development. Hence, our data provided novel insights into PIWI/piRNA-mediated gene regulation and their function in pancreatic cancer. Since PIWI proteins and piRNA predominately express in germline and cancer cells, our study provided novel therapeutic strategy for pancreatic cancer treatment.

Erythropoietin-producing hepatocellular receptor A7 restrains estrogen negative feedback of luteinizing hormone via ephrin A5 in the hypothalamus of female rats.

We have previously shown that systemic injection of erythropoietin-producing hepatocellular receptor A7 (EPHA7)-Fc raises serum luteinizing hormone (LH) levels before ovulation in female rats, indicating the induction of EPHA7 in ovulation. In this study, we aimed to identify the mechanism and hypothalamus-pituitary-ovary (HPO) axis level underlying the promotion of LH secretion by EPHA7. Using an ovariectomized (OVX) rat model, in conjunction with low-dose 17ß-estradiol (E2) treatment, we investigated the association between EPHA7-ephrin (EFN)A5 signaling and E2 negative feedback. Various rat models (OVX, E2-treated OVX, and abarelix treated) were injected with the recombinant EPHA7-Fc protein through the caudal vein to investigate the molecular mechanism underlying the promotion of LH secretion by EPHA7. Efna5 was observed strongly expressed in the arcuate nucleus of the female rat by using RNAscope in situ hybridization. Our results indicated that E2, combined with estrogen receptor (ER)α, but not ERß, inhibited Efna5 and gonadotropin-releasing hormone 1 (Gnrh1) expressions in the hypothalamus. In addition, the systemic administration of EPHA7-Fc restrained the inhibition of Efna5 and Gnrh1 by E2, resulting in increased Efna5 and Gnrh1 expressions in the hypothalamus as well as increased serum LH levels. Collectively, our findings demonstrated the involvement of EPHA7-EFNA5 signaling in the regulation of LH and the E2 negative feedback pathway in the hypothalamus, highlighting the functional role of EPHA7 in female reproduction.

MiR-645 promotes invasiveness, metastasis and tumor growth in colorectal cancer by targeting EFNA5.

MicroRNA-645 (miR-645) has been implicated in numerous types of human cancers including colon cancer. However, the effects and mechanisms of action of miR-645 dysregulation on the growth and malignancy of colorectal cancer (CRC) remain unclear. In this study, we demonstrated that miR-645 knockdown significantly diminished CRC cell migration and invasion and repressed epithelial-mesenchymal transition (EMT). Conversely, miR-645 overexpression enhanced CRC cell migration, invasion, and EMT. In vivo assays confirmed that miR-645 knockdown substantially reduced CRC growth and metastasis. Regarding the mechanism, ephrin-A5 (EFNA5) was identified as a direct target gene of miR-645. MiR-645 specifically targeted the 3'-untranslated region of EFNA5 mRNA and hindered its expression. EFNA5 knockdown attenuated the effects of miR-645 knockdown on CRC cell migration and invasion. Additionally, we noted a statistically significant inverse correlation between EFNA5 mRNA and miR-645 levels in tumors from 28 patients with CRC. Hence, miR-645 acts as an oncogenic miRNA that may increase CRC cell migration, invasiveness, and metastasis by targeting EFNA5.

Ephrin-A2 affects wound healing and scarring in a murine model of excisional injury.

Ephrin ligand/Eph receptor signaling is important in both tissue development and homeostasis. There is increasing evidence that Ephrin/Eph signaling is important in the skin, involved in hair follicle cycling, epidermal differentiation, cutaneous innervation and skin cancer. However, there is currently limited information on the role of Ephrin/Eph signaling in cutaneous wound healing. Here we report the effects of the Ephrin-A2 and A5 ligands on wound healing. Using Ephrin-A2-/-, Ephrin-A5-/- and Ephrin-A2A5-/- transgenic mice, in vitro wound healing assays were conducted using isolated keratinocytes and fibroblasts. Ephrin-A2-/-, Ephrin-A2A5-/- and wild type mice with excisional wounds were used to analyze the impact of these ligands on wound closure, scar outcome, collagen orientation and re-innervation in vivo. The absence of the Ephrin-A2 and A5 ligands did not have any effect on dermal fibroblast proliferation or on fibroblast or keratinocyte migration. The loss of Ephrin-A2 and A5 ligands did not impact on the rate of wound closure or re-innervation after injury. However, changes in the gross morphology of the healed scar and in collagen histology of the scar dermis were observed in transgenic mice. Therefore Ephrin-A2 and A5 ligands may play an important role in final scar appearance associated with collagen deposition and structure.

Epha2 and Efna5 participate in lens cell pattern-formation.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

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.

Lhx2 Expression in Postmitotic Cortical Neurons Initiates Assembly of the Thalamocortical Somatosensory Circuit.

Cortical neurons must be specified and make the correct connections during development. Here, we examine a mechanism initiating neuronal circuit formation in the barrel cortex, a circuit comprising thalamocortical axons (TCAs) and layer 4 (L4) neurons. When Lhx2 is selectively deleted in postmitotic cortical neurons using conditional knockout (cKO) mice, L4 neurons in the barrel cortex are initially specified but fail to form cellular barrels or develop polarized dendrites. In Lhx2 cKO mice, TCAs from the thalamic ventral posterior nucleus reach the barrel cortex but fail to further arborize to form barrels. Several activity-regulated genes and genes involved in regulating barrel formation are downregulated in the Lhx2 cKO somatosensory cortex. Among them, Btbd3, an activity-regulated gene controlling dendritic development, is a direct downstream target of Lhx2. We find that Lhx2 confers neuronal competency for activity-dependent dendritic development in L4 neurons by inducing the expression of Btbd3.

Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse.

Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo.

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.

OncomiR miR-96 and miR-182 promote cell proliferation and invasion through targeting ephrinA5 in hepatocellular carcinoma.

EphrinA5, a member of the ephrinA subclass, is downregulated in hepatocellular carcinoma (HCC) and acts as a tumor suppressor. However, the upstream regulation mechanism of ephrinA5 remains unclear. In this study, we tried to identify and characterize the roles of miR-96 and miR-182 in the regulation of ephrinA5 expression in HCC. The expression levels of miR-96 and miR-182 were examined in 47 paired HCC and para-tumoral liver tissues using quantitative real-time RT-PCR. The luciferase reporter assay and western blotting were employed to dissect the association between miR-96/182 and ephrinA5 expression. Moreover, cells were treated with synthetic miR-96/182 precursors and inhibitors to assess their effects on HCC cell growth and migration. It was found that both miR-96 and miR-182 were upregulated in HCC compared to para-tumoral normal tissues. The expression of miR-96 and miR-182 was inversely associated with ephrinA5 protein levels. Furthermore, both miR-96 and miR-182 directly targeted the 3'UTR of the ephrinA5 mRNA and suppressed protein translation. The suppression of miR-96 and miR-182 led to reduced HCC cell proliferation and migration by negatively regulating ephrinA5 expression. In conclusion, miR-96 and miR-182 may act as oncomiRs in HCC by suppressing the expression of ephrinA5 and may play important roles in hepatocarcinogenesis. © 2015 Wiley Periodicals, Inc.

Rapid Changes in Cortical and Subcortical Brain Regions after Early Bilateral Enucleation in the Mouse.

Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic ephrin A5 expression in the same location ten days later at postnatal day (P) 10. A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on (second postnatal week). With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.

Effects of altered ephrin-A5 and EphA4/EphA7 expression on tumor growth in a medulloblastoma mouse model.

BACKGROUND: Members of the Eph/ephrin gene families act as key regulators of cerebellar development during embryogenesis. Aberrant signaling of Eph family of receptor tyrosine kinases and their ephrin ligands has also been implicated in human cancers. Medulloblastoma is an aggressive primitive neuroectodermal tumor that originates from granule neuron precursors in the cerebellum. Previous studies have suggested a role for the ephrin-A5 ligand and its receptors, EphA4 and EphA7, in granule cell-precursor formation and in guiding cell migration. In the present study, we investigated the effects of genetic loss of ephrin-A5, EphA4, and EphA7 on the spatiotemporal development of medulloblastoma tumors in the context of the smoothened transgenic mouse model system. FINDINGS: Radiographic magnetic resonance imaging (MRI) was performed to monitor tumor growth in a genetically engineered mouse model of medulloblastoma. Tumor tissue was harvested to determine changes in the expression of phosphorylated Akt by Western blotting. This helped to establish a correlation between genotype and/or tumor size and survival. Our in vivo data establish that in ND2-SmoA1 transgenic mice, the homozygous deletion of ephrin-A5 resulted in a consistent pattern of tumor growth inhibition compared to their ephrin-A5 wild-type littermate controls, while the loss of EphA4/EphA7 failed to produce consistent effects versus EphA4/EphA7 wild-type mice. A positive correlation was evident between tumor size, p-Akt, and proliferating cell nuclear antigen (PCNA) expression in our transgenic mouse model system, regardless of genotype. CONCLUSIONS: Taken together, our findings underscore the importance of targeting specific members of the Eph/ephrin families in conjunction with the Akt pathway in order to inhibit medulloblastoma tumor growth and progression.

A multigene assay identifying distinct prognostic subtypes of clear cell renal cell carcinoma with differential response to tyrosine kinase inhibition.

Patients with clear cell renal cell carcinoma (ccRCC) have divergent survival outcomes and therapeutic responses, which may be determined by underlying molecular diversity. We aimed to develop a practical molecular assay that can identify subtypes with differential prognosis and response to targeted therapy. Whole-genome expression analysis of formalin-fixed paraffin-embedded (FFPE) material from 55 ccRCC patients was performed and two molecular subtypes with differential clinical outcomes were identified by hierarchical clustering. An eight-gene quantitative polymerase chain reaction assay for classification into two subtypes was developed for FFPE material. The primary objective was to assess assay performance by correlating ccRCC prognostic subtypes to cancer-specific survival (CSS) and, for patients receiving targeted therapy, radiologic response. In three validation cohorts, patients could be distinguished into prognostic subtypes with differential CSS (Singapore General Hospital FFPE cohort: n = 224; p = 1.48 × 10(-8); the Cancer Genome Atlas RNA-Sequencing cohort: n = 419; p = 3.06 × 10(-7); Van Andel Research Institute microarray cohort: n=174; p=0.00743). For 48 patients receiving tyrosine kinase inhibitor (TKI) treatment, the prognostic classification was associated with radiologic response to treatment (p = 5.96 × 10(-4)) and prolonged survival on TKI treatment (p=0.019). The multigene assay can classify ccRCCs into clinical prognostic subtypes, which may be predictive of response in patients receiving TKI therapy.

Mutational screening of EFNA5 in Chinese age-related cataract patients.

BACKGROUND/AIM: In the past few years, Ephrin-A5 (EFNA5) had been identified to be associated with lens development, but so far no sequence variation in EFNA5 has been reported in humans. Therefore, we conduct this study to investigate the EFNA5 genetic variations in Chinese age-related cataract (ARC) patients. METHODS: Sequencing of EFNA5 was performed in 140 sporadic ARC patients and 142 random unrelated healthy subjects. Genomic DNA was extracted from peripheral blood leukocytes. All exons of EFNA5 were sequenced after being amplified by polymerase chain reaction. The functional consequences of the variations were analyzed using PolyPhen2. RESULTS: Three single nucleotide polymorphisms in EFNA5, c.668C>T (rs201008479), c.102C>T (rs199980747) and c.-27C>G (rs200187971), were found in the patients, and none of them presented in the normal controls. Using PolyPhen2, c.668C>T in EFNA5 is predicted to be possibly damaging. CONCLUSIONS: The genetic variations c.668C>T (rs201008479), c.102C>T (rs199980747) and c.-27C>G (rs200187971) may present an additional genetic risk factor for ARC in the Chinese population. This study shows the first cases of these genetic variations in EFNA5 in human beings.

Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5.

Eph receptor tyrosine kinases and their ephrin ligands mediate cell signaling during normal and oncogenic development. Eph signaling is initiated in a multistep process leading to the assembly of higher-order Eph/ephrin clusters that set off bidirectional signaling in interacting cells. Eph and ephrins are divided in two subclasses based on their abilities to bind and activate each other and on sequence conservation. EphA4 is an exception to the general rule because it can be activated by both A- and B-class ephrin ligands. Here we present high-resolution structures of the complete EphA4 ectodomain and its complexes with ephrin-A5. The structures reveal how ligand binding promotes conformational changes in the EphA4 ligand-binding domain allowing the formation of signaling clusters at the sites of cell-cell contact. In addition, the structural data, combined with structure-based mutagenesis, reveal a previously undescribed receptor-receptor interaction between the EphA4 ligand-binding and membrane-proximal fibronectin domains, which is functionally important for efficient receptor activation.

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1.

A previous study showed that the EphA7 receptor regulates apoptotic cell death during early brain development. In this study, we provide evidence that the EphA7 receptor interacts with death receptors such as tumor necrosis factor receptor 1 (TNFR1) to decrease cell viability. We showed that ephrinA5 stimulates EphA7 to activate the TNFR1-mediated apoptotic signaling pathway. In addition, a pull-down assay using biotinylated ephrinA5-Fc revealed that ephrinA5-EphA7 complexes recruit TNFR1 to form a multi-protein complex. Immunocytochemical staining analysis showed that EphA7 was co-localized with TNFR1 on the cell surface when cells were incubated with ephrinA5 at low temperatures. Finally, both the internalization motif and death domain of TNFR1 was important for interacting with an intracytoplasmic region of EphA7; this interaction was essential for inducing the apoptotic signaling cascade. This result suggests that a distinct multi-protein complex comprising ephrinA5, EphA7, and TNFR1 may constitute a platform for inducing caspase-dependent apoptotic cell death.