Eyes Absent Homolog Research Articles

EYA-1 is required for genomic integrity independent of H2AX signalling in Caenorhabditis elegans.

Resolving genomic insults is essential for the survival of any species. In the case of eukaryotes, several pathways comprise the DNA damage repair network, and many components have high evolutionary conservation. These pathways ensure that DNA damage is resolved which prevents disease associated mutations from occurring in a de novo manner. In this study, we investigated the role of the Eyes Absent (EYA) homologue in Caenorhabditis elegans and its role in DNA damage repair. Current understanding of mammalian EYA1 suggests that EYA1 is recruited in response to H2AX signalling to dsDNA breaks. C. elegans do not possess a H2AX homologue, although they do possess homologues of the core DNA damage repair proteins. Due to this, we aimed to determine if eya-1 contributes to DNA damage repair independent of H2AX. We used a putative null mutant for eya-1 in C. elegans and observed that absence of eya-1 results in abnormal chromosome morphology in anaphase embryos, including chromosomal bridges, missegregated chromosomes, and embryos with abnormal nuclei. Additionally, inducing different types of genomic insults, we show that eya-1 mutants are highly sensitive to induction of DNA damage, yet show little change to induced DNA replication stress and display a mortal germline resulting in sterility over successive generations. Collectively, this study suggests that the EYA family of proteins may have a greater involvement in maintaining genomic integrity than previously thought and unveils novel roles of EYA associated DNA damage repair.

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

Tripartite motif containing 69 elicits ERK2-dependent EYA4 turnover to impart pancreatic tumorigenesis.

Eyes absent homologue 4 (EYA4) is silenced in pancreatic ductal adenocarcinoma (PDAC) and functions as a tumor suppressor to restrain PDAC development, albeit the molecular mechanism underlying its downregulation remains enigmatic. Methods: Functional studies were determined by immunohistochemistry of PDAC samples from patients and Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, three-dimensional spheroid culture, flow cytometry, MTT and subcutaneous xenograft experiments. Mechanistical studies were examined by cellular ubiquitination, cycloheximide (CHX) pulse-chase, co-immunoprecipitation, chromatin immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and luciferase reporter assays. Results: We screen E3 ligase that is negatively correlated with EYA4 and uncover a mutually exclusive interaction of tripartite motif containing 69 (TRIM69) with EYA4 in human PDAC. TRIM69 elicits EYA4 polyubiquitylation and turnover independent of P53 and impedes the EYA4-driven deactivation of β-catenin/ID2 cascade, fueling PDAC cell proliferation in vitro and tumor development in mice. Expression of TRIM69 is upregulated in PDAC samples from independent cohorts of patients and the Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+ (KPC) mice, and associated with unfavorable prognosis. Depleting TRIM69 preferentially induces lethality in the EYA4-deficient PDAC cells. We further unearth that ERK2 directly binds to the D-site of mitogen-activated protein kinase (MAPK) docking groove in EYA4 Leu512/514 and phosphorylates EYA4 at Ser37, which is instrumental for EYA4 polyubiquitylation and turnover by TRIM69. Conclusion: Our results define a previously unappreciated role of TRIM69-EYA4 axis in pancreatic tumorigenesis and underscore that targeting TRIM69 might be an effective therapeutic approach for PDAC harboring EYA4 deficiency.

Inhibitors of EYA3 Protein in Ewing Sarcoma.

Objective:Among sarcomas, Ewing sarcoma (EWS) is characterized as a highly malignant type of bone tumor caused by the fusion of EWS RNA Binding Protein-1 (EWSR1)/ Friend leukemia integration 1 (FLI1) genes. The product of fusion gene gives rise to EWSR1/FLI1 which activates the activity of Eyes absent homolog 3 (EYA3) which causes tumor growth and angiogenesis. EYA3 is now considered as a therapeutic drug target for EWS . The study was designed to gather potential inhibitors for the EYA3 target using medicinal compounds. Methods: In this study, we have obtained a list of medicinal compounds from the NuBBE database and downloaded their structural information. Then insilico screening analysis of >2,000 medicinal compounds was performed with PyRX virtual drug screening software to discover potential inhibitors for the treatment of EWS. Results:Our investigation revealed that Sorbifolin and 1,7-Dihydroxy-3-methylanthracene-9.10-dione show interactive affinity for EYA3 active residues. Moreover, these compounds have adequate toxicity, can induce cytotoxicity in EWS cells, and are capable of regulating the expression of genes activated by EWSR1/FLI1. Conclusion:Our study concluded that Sorbifolin and 1,7-Dihydroxy-3-methylanthracene-9.10-dione are promising drug candidates for the treatment of EWS and should be further subjected to invitro testing.

The deubiquitinase OTUB1 fosters papillary thyroid carcinoma growth through EYA1 stabilization.

Deubiquitinating enzyme OTU domain‐containing ubiquitin aldehyde‐binding proteins 1 (OTUB1) has been shown to have an essential role in multiple carcinomas. However, the function of OTUB1 in papillary thyroid cancer (PTC) and the underlying mechanisms regulating PTC cells proliferation remain poorly understood. In this study, OTUB1 was significantly upregulated in papillary thyroid carcinoma tissues and cells. Through in vitro and in vivo experiments, knockdown of OTUB1 suppressed PTC cells growth whereas OTUB1 overexpression enhanced the proliferation ability of PTC cells. Moreover, the eyes absent homologue 1 (EYA1) was recognized as a potential target of OTUB1 through mass spectrometry analysis, and we further verified that EYA1 protein level was positively correlated with OTUB1 expression in PTC cells and clinical samples. Mechanistically, OTUB1 could interact with EYA1 directly and deubiquitinate EYA1 to stabilize it. At last, EYA1 was found to play an essential role in OTUB1‐derived PTC cells growth. Overall, our investigation reveals that OTUB1 is a previously unrecognized oncogenic factor in PTC cells proliferation and suggests that OTUB1 might be a novel therapeutic target in PTC.

Two naturally derived small molecules disrupt the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) interaction to inhibit colorectal cancer cell growth.

Background:Emerging evidence indicates that the sineoculis homeobox homolog 1−eyes absent homolog 1 (SIX1–EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the SIX1–EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of SIX1–EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the SIX1–EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.Methods:Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n = 24). In vivo immunoprecipitation and in vitro pulldown assays were carried out to determine SIX1–EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted SIX1–EYA1 interaction. CRC cells harboring different levels of SIX1/EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.Results:Both SIX1 and EYA1 were overexpressed in CRC cancerous tissues (for SIX1, 7.47 ± 3.54 vs.1.88 ± 0.35, t = 4.92, P = 0.008; for EYA1, 7.61 ± 2.03 vs. 2.22 ± 0.45, t = 6.73, P = 0.005). The SIX1/EYA1 complex could mediate the expression of two important genes including cyclin A1 (CCNA1) and transforming growth factor beta 1 (TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both SIX1 and EYA1 could decrease cell proliferation, cell invasion, tumor growth, and in vivo tumor growth (all P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the SIX1–EYA1 interaction with a half-maximal inhibitory concentration (IC50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of CCNA1 and TGFB1 and inhibit the growth of CRC cells in vitro and in vivo.Conclusions:Overexpression of the SIX1/EYA1 complex transactivated the expression of CCNA1 and TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the SIX1–EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.

EYA2 suppresses the progression of hepatocellular carcinoma via SOCS3-mediated blockade of JAK/STAT signaling

BackgroundSomatic mutations are involved in hepatocellular carcinoma (HCC) progression, but the genetic mechanism associated to hepatocarcinogenesis remains poorly understood. We report that Eyes absent homolog 2 (EYA2) suppresses the HCC progression, while EYA2(A510E) mutation identified by exome sequencing attenuates the tumor-inhibiting effect of EYA2.MethodsWhole-exome sequencing was performed on six pairs of human HCC primary tumors and matched adjacent tissues. Focusing on EYA2, expression level of EYA2 in human HCC samples was evaluated by quantitative real-time PCR, western blot and immunohistochemistry. Loss- and gain-of-function studies, hepatocyte-specific deletion of EYA2 (Eya2−/−) in mice and RNA sequencing analysis were used to explore the functional effect and mechanism of EYA2 on HCC cell growth and metastasis. EYA2 methylation status was evaluated using Sequenom MassARRAY and publicly available data analysis.ResultsA new somatic mutation p.Ala510Glu of EYA2 was identified in HCC tissues. The expression of EYA2 was down-regulated in HCC and associated with tumor size (P = 0.001), Barcelona Clinic Liver Cancer stage (P = 0.016) and tumor differentiation (P = 0.048). High level of EYA2 was correlated with a favorable prognosis in HCC patients (P = 0.003). Results from loss-of-function and gain-of-function experiments suggested that knockdown of EYA2 enhanced, while overexpression of EYA2 attenuated, the proliferation, clone formation, invasion, and migration of HCC cells in vitro. Delivery of EYA2 gene had a therapeutic effect on inhibition of orthotopic liver tumor in nude mice. However, EYA2(A510E) mutation led to protein degradation by unfolded protein response, thus weakening the inhibitory function of EYA2. Hepatocyte-specific deletion of EYA2 in mice dramatically promoted diethylnitrosamine-induced HCC development. EYA2 was also down-regulated in HCC by aberrant CpG methylation. Mechanically, EYA2 combined with DACH1 to transcriptionally regulate SOCS3 expression, thus suppressing the progression of HCC via SOCS3-mediated blockade of the JAK/STAT signaling pathway.ConclusionsIn our study, we identified and validated EYA2 as a tumor suppressor gene in HCC, providing a new insight into HCC pathogenesis.

Two Isoforms of Eya3 Influence Muscle Cell Differentiation and Become Dysregulated in Striated Muscle Diseases

Alternative splicing is a ubiquitous gene regulatory mechanism by which diverse transcripts are derived from a finite genome. During development, complex splicing transitions occur, leading to varied transcript profiles in fetal and adult tissues. Like gene expression, splicing often becomes dysregulated in cancer, neurological, and muscular diseases. One barrier to therapeutic targeting of alternative splicing is that the function of splice isoforms, both physiologically and pathologically, are vastly understudied. Here, we begin to address this knowledge gap by describing an evolutionarily conserved alternative splicing event in eyes absent homolog 3 (Eya3) gene for the first time. The short isoform of Eya3 is a dual threonine- and tyrosine-phosphatase that is known to promote cell growth and chemotherapeutic resistance. By analyzing RNA-sequencing data from various mouse tissues, we identified the long isoform of Eya3, which contains an additional 138nt, representing a 46 amino acid in-frame insertion immediately following the threonine phosphatase domain. Using reverse transcription-PCR (RT-PCR) of mouse, human, and rabbit tissues, we discovered that Eya3 splicing is tissue-specific, with the long isoform expressed exclusively in the heart, skeletal muscle, and brain. Additionally, we found that during heart and skeletal muscle development, expression of the long Eya3 isoform gradually increases relative to the expression of the short isoform. In myotonic dystrophy type 1 patient hearts, and failing mouse hearts, the short Eya3 isoform is re-expressed, illustrating that Eya3 splicing is dysregulated in striated muscle diseases. To understand Eya3 splicing in further molecular detail, we utilized the well-established C2C12 murine myoblast cell line. Differentiation of myoblasts to myotubes robustly recapitulates the Eya3 splicing transition observed during tissue development, making them an appropriate cell culture system for our work. Depletion of Eya3 via siRNA transfection inhibited myoblast proliferation and fusion. Interestingly, blocking the endogenous Eya3 alternative splicing transition with morpholino antisense oligonucleotides also suppressed normal differentiation as determined by immunohistochemical staining and confocal microscopy. These results indicate that Eya3, and its splice isoforms, play an important role in early muscle development. In summary, our studies uncover a developmentally-regulated, tissue-specific, and disease-relevant splicing event in Eya3. Moving forward, we seek to determine the molecular mechanism by which Eya3 isoforms regulate normal muscle development and to evaluate Eya3 as a potential therapeutic target.

MicroRNA-219 inhibits cell viability and metastasis in papillary thyroid carcinoma by targeting EYA2.

In recent years, the incidence of papillary thyroid carcinoma (PTC) has increased. Many microRNAs (miRNAs) have been found to regulate PTC progression. However, the regulatory mechanism of miR-219 remains unclear in PTC. Therefore, the purpose of this study is to explore the function of miR-219 in PTC. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot analysis were used to detect the expression of miR-219 and eyes absent homologue 2 (EYA2). The function of miR-219 was investigated by methyl thiazolyl tetrazolium (MTT) and transwell assays. The relationship between miR-219 and EYA2 was confirmed by Dual-Luciferase reporter assay. MiR-219 expression was reduced and was associated with TNM stage and lymph node metastases in PTC patients. Functionally, overexpression of miR-219 restrained the viability and metastasis of PTC cells. In addition, miR-219 induced apoptosis and blocked EMT in PTC cells. Furthermore, miR-219 was confirmed to directly target EYA2 and inhibited its expression in PTC. More importantly, the upregulation of EYA2 impaired the inhibitory effect of miR-219 in PTC. MiR-219 inhibits the viability and metastasis of PTC cells by downregulating EYA2.

Co-transplantation with adipose-derived cells to improve parathyroid transplantation in a mice model

BackgroundAccidentally removed parathyroid glands are still challenging in neck surgery, leading to hypoparathyroidism characterized with abnormally low levels of parathyroid hormone. Parathyroid auto-transplantation is usually applied in compensation. To improve the efficiency of parathyroid transplantation, we introduced a method by co-transplanting with adipose-derived cells, including stromal vascular fractions (SVFs) and adipose-derived stem cells (ADSCs), and investigated the underlying molecular mechanisms involved in parathyroid transplantation survival.MethodsRat and human parathyroid tissues were transplanted into nude mice as parathyroid transplantation model to examine the effects of SVFs and ADSCs on grafts angiogenesis and survival rates, including blood vessel assembly and parathyroid hormone levels. Several angiogenic factors, such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF) 2, were assessed in parathyroid grafts. The effects of hypoxia were investigated on ADSCs. The modulatory roles of the eyes absent homolog 1 (EYA1), which is vital in parathyroid development, was also investigated on angiogenic factor production and secretion by ADSCs. All experimental data were statistically processed. Student’s t test was used to assess significant differences between 2 groups. For multiple comparisons with additional interventions, two-way ANOVA followed by Tukey’s post hoc test was performed. P < 0.05 was considered as significant.ResultsSVFs improve rat parathyroid transplantation survival and blood vessel assembly, as well as FGF2 and VEGF-A expression levels in parathyroid transplantation mice. Functional human parathyroid grafts have higher microvessel density and increased VEGF-A expression. The supernatant of ADSCs induced tubule formation and migration of human endothelial cells in vitro. Hypoxia had no effect on proliferation and apoptosis of human ADSCs but induced higher angiogenic factor levels of VEGF-A and FGF2, modulated by EYA1, which was confirmed by parathyroid glands transplantation in mice.ConclusionsAdipose-derived cells, including ADSCs and SVFs, improve parathyroid transplantation survival via promoting angiogenesis through EYA1-regulating angiogenetic factors in vitro and in vivo. Our studies proved an effective method to improve the parathyroid autotransplantation, which is promising for clinical patients with hypoparathyroidism when parathyroid glands were accidentally injured, removed, or devascularized.

LINC00511 as a ceRNA promotes cell malignant behaviors and correlates with prognosis of hepatocellular carcinoma patients by modulating miR-195/EYA1 axis

BackgroundRecently, a growing number of reports indicated that long non-coding RNAs (lncRNAs) were involved in the development of various cancers. However, the performance of LINC00511 is still limited in hepatocellular carcinoma (HCC). Thus, we attempted to assess the effect of LINC00511 and underlying mechanism in HCC progression. MethodsTCGA and GEO database acted as supporters to provide us clinical samples data. Overall survival (OS) analyses were plotted using Kaplan-Meier method. Five cell lines were utilized to detect LINC00511 expression level and Cell Counting Kit-8 (CCK-8), colony formation and transwell assays were conducted to examine the effects on cell behaviors. The correlations between LINC00511 and miR-195 or eyes absent homolog 1 (EYA1) were confirmed by luciferase reporter assay. Quantitative real-time PCR and western blotting were fulfilled to ascertain the mRNA and protein expression levels. ResultsIn this study, we found that LINC00511 was high-regulated in HCC tissue samples and cell lines, which might be linked with unfavorable prognosis of HCC patients and clinical parameters. Loss-of-function experiments determined that LINC00511 deficiency inhibited cell proliferation, colony formation and invasive activity in HepG2 cells, while gain-of-function experiments showed the counter impacts in Huh7 cells. Bioinformatics tools and luciferase reporter assays revealed that LINC00511 may act as a competing endogenous RNA (ceRNA) for miR-195 and positively correlate with EYA1, which was reinforced by rescue experiments. ConclusionTaken together, these findings indicated that LINC00511 interacted with EYA1 promoted HCC development via mediating miR-195, proposing a promising therapeutic biomarker for HCC diagnosis and prognosis.

EYA2 Correlates With Clinico-Pathological Features of Breast Cancer, Promotes Tumor Proliferation, and Predicts Poor Survival.

Eyes absent homolog 2 (EYA2), a transcriptional activator, is pivotal for organ development, but aberrant regulation of EYA2 has been reported in multiple human tumors. However, the role of EYA2 in breast cancer is still lack of full understanding. To explore the biological significance of EYA2 in breast cancer, we conducted data analysis on public breast cancer datasets, and performed immunohistochemistry (IHC) analysis, colony-forming unit assays, EdU assay, western blotting, and immunofluorescence (IF). Meta-analysis showed that EYA2 mRNA expression was correlated with tumor grade, the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). IHC analysis displayed that EYA2 protein abundance was inversely associated with the status of ER and PR, and enriched in triple-negative breast cancer in comparison with luminal-type tumors. Additionally, correlation analysis reflected that EYA2 mRNA was negatively correlated with luminal markers, and positively associated with markers of basal cells, epithelial-mesenchymal transition and cancer stem cells. Clone-forming assay and EdU experiment showed that EYA2 overexpression enhanced proliferation of breast cancer cells. Results from western blotting and IF displayed that overexpression of EYA2 up-regulated the protein abundance of proliferation markers. Importantly, survival analysis indicated that higher EYA2 mRNA level predicted worse overall survival, relapse-free survival and metastasis-free survival among whole enrolled breast cancer patients. Collectively, EYA2 was closely correlated with clinico-pathological characteristics, and served as a proliferation stimulator for breast cancer cells and an unfavorable prognostic element for breast cancer patients, suggesting that EYA2 is involved in the progression of breast carcinoma.

Lineage context switches the function of a C. elegans Pax6 homolog in determining a neuronal fate.

The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoter-selection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner. This article has an associated 'The people behind the papers' interview.

Alteration of gut microbiota-associated epitopes in children with autism spectrum disorders

BackgroundAutism spectrum disorder (ASD) affects 1% of children and has no cure. Gastrointestinal (GI) problems are common in children with ASD, and although gut microbiota is known to play an important role in ASD through the gut–brain axis, the specific mechanism is unknown. Recent evidence suggests that gut microbiota may participate in the pathogenesis of ASD through immune- and inflammation-mediated pathways. Here, we identified potentially immunogenic epitopes derived from gut microbiota in stool samples from ASD children with and without GI problems and typically developing (TD) children. MethodsCandidate gut microbiota-associated epitopes (MEs) were identified by blast shotgun metagenomic sequencing of fecal samples from 43 ASD children (19 with and 24 without GI involvement) and 31 sex- and age-matched typically developing (TD) children. Potentially immunogenic epitopes were screened against a predictive human Immune Epitope Database. The composition and abundance of candidate MEs were compared between the three groups of children. ResultsMEs identified in ASD children with GI problems were significantly more diverse than those in TD children. ME composition could discriminate between the three groups of children. We identified 34 MEs that were significantly more or less abundant in ASD children than TD children, most (29/34) of the differences in MEs were reduced in ASD and associated with abnormal gut IgA level and altered gut microbiota composition, these MEs were limited effected by clinical factors such as age, gender, and GI problems, of which eleven MEs were pathogenic microorganisms peptides with strong T or B cell response, nine MEs showed high homology to peptides from human self proteins associated with autoimmune disease occurrence, eliciting immune attack against hematopoietic stem cells and inhibition antigen binding. We also found that the abundance of five MEs were increased in ASD, including three human self proteins, gap junction alpha-1 (GJA1), paired box protein Pax-3 (PAX3) and eyes absent homolog 1 isoform 4 (EYA1) which associated with cancer, and a ME with homology to a Listeriolysin O peptide from the pathogenic bacterium Listeria monocytogenes was significantly increased in ASD children compared with TD children. ConclusionsOur findings demonstrate the abnormal of MEs composition in the gut of children with ASD, moreover, the abnormality in MEs composition was associated with abnormal gut IgA levels and altered gut microbiota composition, this abnormality also suggests that there may be abnormalities in intestinal immunity in children with ASD; In all, thirty-four MEs identified were potential biomarker of ASD, and alterations in MEs may contribute to abnormalities in gut immunity and/or homeostasis in ASD children. Further study of the MEs identified here may advance our understanding of the pathogenesis of ASD.

Eyes absent homologue 2 predicts a favorable prognosis in colorectal cancer.

PurposeEyes absent homologue 2 (EYA2), which functions as a transcription activator and phosphatase, plays an important role in several types of cancer. However, the impact of EYA2 in colorectal cancer (CRC) remains elusive.Patients and methodsWe evaluated the significance of EYA2 expression in the development and progression of CRC in a large cohort, including 922 CRC cases. EYA2 protein expression was determined via immunohistochemistry in colorectal tissues. The correlation between EYA2 expression and CRC occurrence was investigated in tumor tissue and the adjacent normal tissues. Factors contributing to CRC prognosis were evaluated using Kaplan–Meier and Cox model analyses.ResultsEYA2 expression was progressively lower in the adjacent normal tissue, adenomas, primary tumor and the metastatic CRC (all P<0.05). Furthermore, EYA2 expression had significant associations with disease stage, differentiation grade, and number of resected lymph nodes (all P<0.001). Compared with patients with EYA2-high tumors, those with EYA2-low tumors had shorter disease-free survival (hazard ratio [HR], 2.347; 95% CI, 1.665–3.308) and disease-specific survival (HR, 3.560; 95% CI, 2.055–6.167) in multivariate Cox analysis, after adjusting confounding factors such as tumor-node-metastasis stage and grade. In particular, patients with stage II or III EYA2-low CRC might be harmed by postoperative chemotherapy.ConclusionEYA2 expression was generally reduced in CRC. Higher EYA2 expression can predict a more favorable prognosis for CRC.

EYA4 inhibits hepatocellular carcinoma growth and invasion by suppressing NF-\u03baB-dependent RAP1 transactivation

BackgroundOur previous studies demonstrated that eyes absent homolog 4 (EYA4), a member of the eye development-related EYA family in Drosophila, is frequently methylated and silenced in hepatocellular carcinoma (HCC) specimens and associated with shorter survival. The current work aimed to explore the mechanisms through which EYA4 functions as a tumor suppressor in HCC.MethodsStable EYA4-expressing plasmid (pEYA4) transfectants of the human HCC cell lines Huh-7 and PLC/PRF/5 (PLC) were established. Xenografts tumors were established via subcutaneous injection of the stable transfectants into BALB/c nude mice. Tissue samples were obtained from 75 pathologically diagnosed HCC patients. Quantitative real-time polymerase chain reaction, Western blotting and immunohistochemistry were performed to determine the expression of EYA4 in cell lines, xenografts and clinical specimens. The cell proliferation, colony formation, invasiveness and tumor formation of stable transfectants were studied. A gene expression microarray was utilized to screen genes regulated by EYA4 expression. The effect of EYA4 on nuclear factor-κB (NF-κB)/RAS-related protein 1 (RAP1) signaling was demonstrated through the co-transfection of pEYA4 and Flag-tagged RAS-related protein 1A gene-expressing plasmid (Flag-RAP1A), functional studies, chromatin immunoprecipitation, immunofluorescence staining and cellular ubiquitination assay.ResultsThe restoration of EYA4 expression in HCC cell lines suppressed cell proliferation, inhibited clonogenic outgrowth, reduced cell invasion and restrained xenograft tumor growth, and Flag-RAP1A reversed the suppressive effects of pEYA4 in vitro. Activation of NF-κB with tumor necrosis factor-α (TNF-α) increased the binding of p65 to the RAP1A gene promoter and up-regulated RAP1 protein expression. The inhibition of NF-κB with BAY 11-7085 and p65 siRNA successfully blocked TNF-α-induced RAP1 up-regulation. EYA4 antagonized the TNF-α-induced phosphorylation and ubiquitination of inhibitor of NF-κBα (IκBα) as well as the nuclear translocation and transactivation of p65, resulting in repressed NF-κB activity and RAP1 expression. Blocking the serine/threonine phosphatase activity of EYA4 with calyculin A notably abrogated its suppressive effect on NF-κB activity. In addition, EYA4 expression was inversely correlated with IκBα/RAP1 activity in clinical HCC specimens.ConclusionOur findings provide a functional and mechanistic basis for identifying EYA4 as a bona fide tumor suppressor that disrupts aberrant activation of the NF-κB/RAP1 signaling pathway and thus orchestrates a physiological impediment to HCC growth and invasion.

EYA1 promotes tumor angiogenesis by activating the PI3K pathway in colorectal cancer

Blood vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as vascular endothelial growth factor(VEGF) to induce angiogenesis, thereby promoting metastasis. Here, we report that The Drosophila Eyes Absent Homologue 1 (EYA1), which is overexpressed in colorectal tumor cells, can promote colorectal tumor angiogenesis by coordinating with the hypoxia-inducible factor 1 (HIF-1α) to increase the expression of VEGF-A. Moreover, data indicated that the enhancement of HIF-1α expression by Eya1 depended on its ability to activate the phosphatidylinositol 3-kinase (PI3K) signaling pathways to increase the phosphorylation of AKT subunits. Overexpression of Eya1 increased tumor angiogenesis in vivo and in vitro. Our study suggested that Eya1 is essential in regulating cancer cell-mediated angiogenesis and contributes to tumor growth, and that Eya1 provides a potential and specific target for new anti-angiogenesis drug development.

EYA4 Promotes Cell Proliferation Through Downregulation of p27Kip1 in Glioma

Background/Aims: Accumulating evidence suggests that Eyes Absent Homologue 4 (EYA4) plays an important role in tumorigenesis and progression of various cancers. However, the role of EYA4 in glioma development is still unclear. Methods: The expression of EYA4 was examined in glioma tissues by immunohistochemistry. Cell viability and apoptosis were analyzed by CCK-8, BrdU assay, and flow cytometry. Results: We found that EYA4 was upregulated in glioma, and its expression was positively correlated with advanced tumor stage. Moreover, higher expression of EYA4 predicted a worse overall survival in patients with glioma. Forced overexpression of EYA4 enhanced glioma cell proliferation, and EYA4 suppressed the expression of p27Kip1 directly in these cells. Furthermore, Six1 was required for EYA4 to suppress the expression of p27Kip1 in glioma. Conclusion: Together, we demonstrate that EYA4 promotes cell proliferation by directly suppressing the expression of p27Kip1 in glioma.

Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing.

Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3, and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.

EYA4 gene functions as a prognostic marker and inhibits the growth of intrahepatic cholangiocarcinoma.

BackgroundThe molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma (ICC) have not been well documented. The purpose of this study was to investigate the prognostic value of the eyes absent homolog 4 (EYA4) gene in ICC and its biological effects on ICC growth in vitro and in vivo.MethodsOne hundred twelve patients with ICC who underwent hepatectomy were enrolled in the study. EYA4 mRNA and EYA4 protein levels in ICC and adjacent non-tumoral tissues were evaluated using real-time quantitative polymerase chain reaction and immunohistochemical staining, respectively. EYA4 protein levels in ICC cells were determined using western blot analysis. The associations between EYA4 expression and clinicopathologic features of ICC were analyzed. To identify independent prognostic factors, univariate and multivariate analyses were performed. The biological effects of EYA4 on ICC cells were evaluated by establishing stable EYA4-overexpressing transfectants in vitro, and EYA4’s effects on tumor growth were evaluated by intra-tumoral injection of EYA4-expressing plasmids in a NOD/SCID murine model of xenograft tumors.ResultsICC tissues had significantly lower EYA4 mRNA and protein levels compared with adjacent non-tumoral tissues (both P < 0.001). Univariate and multivariate analyses showed that EYA4 protein level, tumor number, adjacent organ invasion, lymph node metastasis, and tumor differentiation were independent prognostic factors for disease-free survival and overall survival (all P < 0.05). In vitro, EYA4 overexpression inhibited tumor cell growth, foci formation, and cell invasiveness. In vivo, intra-tumoral injection of EYA4-expressing plasmids significantly inhibited ICC growth in the murine xenograft model compared with the control group (P < 0.05).ConclusionEYA4 gene functioned as a molecular prognostic marker in ICC, and its overexpression inhibited tumor growth in vitro and in vivo.