YWHAZ Research Articles

Antifreeze protein type I in the vitrification solution improves the cryopreservation of immature cat oocytes

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 μg/mL (G0, 0 μM); 0.5 μg/mL (G0.5, 0.15 μM), or 1 μg/mL (G1, 0.3 μM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 μg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.

Time- and Region-Specific Selection of Reference Genes in the Rat Brain in the Lithium-Pilocarpine Model of Acquired Temporal Lobe Epilepsy.

Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium-pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium-pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.

CSF 14–3-3 zeta(ζ) isoform is associated with tau pathology and cognitive decline in Alzheimer's disease

14–3-3 is a family of conserved proteins that consist of seven isoforms which are highly expressed in the brain, and 14–3-3 zeta(ζ) is one of the isoforms encoded by the YWHAZ gene. Previous studies demonstrated that 14–3-3ζ is deposited in the neurofibrillary tangles of Alzheimer's disease (AD) brains, and that 14–3-3ζ interacts with tau from the purified neurofibrillary tangles of AD brain extract. The present study examined the cerebrospinal fluid (CSF) 14–3-3ζ levels of 719 participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI), including cognitively normal (CN) participants, patients with mild cognitive impairment (MCI) and patients with AD dementia, and aimed to identify whether CSF 14–3-3ζ is associated with tau pathology. CSF 14–3-3ζ levels were increased in AD, and particularly elevated among tau pathology positive individuals. CSF 14–3-3ζ levels were associated with CSF phosphorylated tau 181 (p-tau) (r = 0.741, P < 0.001) and plasma p-tau (r = 0.293, P < 0.001), which are fluid biomarkers of tau pathology, and could predict tau pathology positive status with high accuracy (area under the receiver operating characteristic curve [AUC], 0.891). CSF 14–3-3ζ levels were also correlated to synaptic biomarker CSF GAP-43 (r = 0.609, P < 0.001) and neuroinflammatory biomarker CSF sTREM-2 (r = 0.507, P < 0.001). High CSF 14–3-3ζ levels at baseline were associated with progressive decline of cognitive function and neuroimaging findings during follow up. In conclusion, this study suggests that CSF 14–3-3ζ is a potential biomarker of AD that may be useful in clinical practice.

Analyzing the Impact of Diesel Exhaust Particles on Lung Fibrosis Using Dual PCR Array and Proteomics: YWHAZ Signaling.

Air pollutants are associated with exacerbations of asthma, chronic bronchitis, and airway inflammation. Diesel exhaust particles (DEPs) can induce and worsen lung diseases. However, there are insufficient data to guide polymerase chain reaction (PCR) array proteomics studies regarding the impacts of DEPs on respiratory diseases. This study was performed to identify genes and proteins expressed in normal human bronchial epithelial (NHBE) cells. MicroRNAs (miRNAs) and proteins expressed in NHBE cells exposed to DEPs at 1 μg/cm2 for 8 h and 24 h were identified using PCR array analysis and 2D PAGE/LC-MS/MS, respectively. YWHAZ gene expression was estimated using PCR, immunoblotting, and immunohistochemical analyses. Genes discovered through an overlap analysis were validated in DEP-exposed mice. Proteomics approaches showed that exposing NHBE cells to DEPs led to changes in 32 protein spots. A transcriptomics PCR array analysis showed that 6 of 84 miRNAs were downregulated in the DEP exposure groups compared to controls. The mRNA and protein expression levels of YWHAZ, β-catenin, vimentin, and TGF-β were increased in DEP-treated NHBE cells and DEP-exposed mice. Lung fibrosis was increased in mice exposed to DEPs. Our combined PCR array-omics analysis demonstrated that DEPs can induce airway inflammation and lead to lung fibrosis through changes in the expression levels of YWHAZ, β-catenin, vimentin, and TGF-β. These findings suggest that dual approaches can help to identify biomarkers and therapeutic targets involved in pollutant-related respiratory diseases.

Lipid modulation during IVM increases the metabolism and improves the cryosurvival of cat oocytes

This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FSK), l-carnitine (LC)]. Subsequently, both the CONTROL group and MIX 18 (CLA+FSK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen 19 species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, 20 PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and 21 post-warming viability assessment. When not using any lipid modulator, an increase (P < 0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P < 0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P > 0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A U vs 1.19 ± 0.14 A U). Although ROS and GSH levels were higher (P < 0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P < 0.05) in the latter (C:1.00 ± 0.20b vs M:0.26 ± 0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P < 0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P < 0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.

14-3-3ζ Pseudogenes: Physiologically and powerful evolutionary forces in the genome

Abstract Contrary to the rigid dogma ruling out the phenotypic relevance of pseudogenes, recent RNA-seq and meta-analyses indicate that 10% of all pseudogenes are transcribed, and 40% of those pseudogenes are translated into protein. This investigation unpackages the 14-3-3ζ (YWHAZ) gene, which has been shown to regulate cytokine signaling, induce antibody response, and reduce inflammation in rats with rheumatoid arthritis. The original YWHAZ gene is located on the 8 thhuman chromosome. Our detailed investigation in the human genome shows that there are ten pseudogenes of YWHAZ. Located across the human genome, these pseudogenes display unique properties, such as having allelic SNPs for Body Mass Index, Prostatic Neoplasms, and Macular Degeneration. Diverse mRNA expression profiles skewed towards reproductive organs and an unusually high transcription rate (9 of 10 being actively transcribed) suggest a strong physiological relevance. Additionally, contrary to the prevailing pseudogene formation theory where all exons are transcribed, YWHAZ pseudogenes have omitted the sixth exon’s UTR region, resulting in a more compact, equally functional gene structure. The evolution of the YWHAZ pseudogene from the parent gene’s framework, combined with some pseudogenes having allelic properties, demonstrates how pseudogenes may be the body’s way to repackage and repurpose parent genes for a more diverse genome and proteome.

YWHAZ variation causes intellectual disability and global developmental delay with brain malformation.

YWHAZ encodes an adapter protein 14-3-3ζ, which is involved in many signaling pathways that control cellular proliferation, migration and differentiation. It has not been definitely correlated to any phenotype in OMIM. To investigate the role of YWHAZ gene in intellectual disability and global developmental delay, we conducted whole-exon sequencing in all of the available members from a large three-generation family and we discovered that a novel variant of the YWHAZ gene was associated with intellectual disability and global developmental delay. This variant is a missense mutation of YWHAZ, p.Lys49Asn/c.147A > T, which was found in all affected members but not found in other unaffected members. We also conducted computational modeling and knockdown/knockin with Drosophila to confirm the role of the YWHAZ variant in intellectual disability. Computational modeling showed that the binding energy was increased in the mutated protein combining with the ligand indicating that the c147A > T variation was a loss-of-function variant. Cognitive defects and mushroom body morphological abnormalities were observed in YWHAZ c.147A > T knockin flies. The YWHAZ knockdown flies also manifested serious cognitive defects with hyperactivity behaviors, which is consistent with the clinical features. Our clinical and experimental results consistently suggested that YWHAZ was a novel intellectual disability pathogenic gene.

P-388 Isolation and characterization of human uterine fluid lavage-derived extracellular vesicles by three different methods for optimal embryo transfer timing discovery

Abstract Study question What is the suitable method for isolating extracellular vesicles (EVs) from a small volume of Uterine Fluid Lavage (UFL)? Summary answer It seems that the qEV column can help in faster, precise, and gentle isolation of EVs from small amounts of UFL. What is known already Several studies have discovered that existing EVs in the UFL, which are informative carriers, reflect cytological and molecular conditions of the uterine environment. The relative amounts of the EV components are different in various physiological situations. So, they are the ideal source for precise molecular analysis of the uterine condition to identify the Window of Implantation (WOI) for appropriate embryo transfer (ET) timing. There is limited information describing the most efficient method for EV isolation from a low-volume sample of biological fluids. In particular, EV separation from some biological fluids such as UFL is more complicated because of the viscosity. Study design, size, duration The study was conducted in two steps; first, the pre-implantation UFL sampling effect was studied on the implantation and clinical pregnancy rates. UFLs were obtained from 20 IVF Frozen Embryo Transfer (FET) patients as the case group. The control group consisted of 20 FET patients without lavage. In the second step, UFLs (n = 30) were collected and pooled. The EVs were extracted by ultracentrifugation (UC), Sucrose cushion (Suc), and Size-exclusion chromatography (by the qEV column) methods. Participants/materials, setting, methods First, UFL was collected two days before frozen blastocyst ET from the case group using an intrauterine insemination catheter attached to a syringe. Then, patients were compared with control at the seventh week. Second, EVs were extracted from pooled UFL samples and characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western Blot (WB). Albumin contamination was detected by SDS-PAGE. Total RNA was extracted, and Real-time PCR was performed for six housekeeping genes. Main results and the role of chance A comparison between case and control groups indicated no significant difference for implantation and clinical pregnancy rates. Moreover, we have confirmed the presence of EVs in UFL that were successfully isolated, by the UC, Suc, the qEV methods. The SEM images showed that the collected vesicles were morphologically spherical. The DLS analysis revealed that the intensity peak of isolated particles with the UC and Suc methods were 281 nm and 216 nm, respectively. Extracted EVs using the qEV technique were smaller and homogenized in size, with an intensity peak of 67.7 nm. SDS-PAGE of EVs showed a weaker albumin band in the qEV column (with a 100 kDa filter) method than the UC, Suc, and qEV column (with a 30 kDa filter) methods. Although, their quantification showed no significant differences. WB analysis indicated common EV markers (D81, CD9, CD63, and TSG101) showed better expression using the qEV extraction technique. But CD9 and TSG101 had lower expression levels in EVs isolated using Suc and UC, respectively. Calnexin was not detected in the isolated EVs. The GAPDH, 18SrRNA, ACTIN, GUSB, HMBS, and YWHAZ genes were detected by real-time PCR in all three EV extraction methods. The 18SrRNA gene showed better CT values. Limitations, reasons for caution Uterine fluid samples collection from qualified study groups and the patient’s follow-up for ensuring successful implantation and clinical pregnancy are time-consuming. The low volume of uterine fluid lavage sample and consequently the low number of extracellular vesicles inside it was also the limitation of this study. Wider implications of the findings Endometrial receptivity assay is difficult to study non-invasively. Herein, endometrial secretome EVs as a minimally invasive source for uterine environment molecular assessment is presented. High-throughput analysis of EV components can offer biomarkers for individual determination of ET timing, providing an additional method to use with transvaginal-ultrasonography for assessing endometrial receptivity. Trial registration number not applicable

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish.

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

P.0241 Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

P.0241 Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

Lentiviral shRNA Construction in Gene Therapy for Knock Down CYPIA1, and YWHAZ gene in Lung Cancer

Lung cancer is a disease that is in the top 10 causes of death globally. It is caused by CYP1A1 and YWHAZ genes expressed in the lungs. One of the solutions offered for lung cancer sufferers is gene therapy. Gene therapy for lung cancer is focused on the knock-down of expressed CYP1A1 and YWHAZ genes. It can be using shRNA transfected into lung cells by lentivirus. The lentivirus envelope is modified using glycoprotein filovirus, one of which is ebolavirus. Lentivirus pseudotyping can recognize certain cells. Especially, Ebolavirus can recognize lung cells. The lentiviral gene is designed to carry the gene of interest (shRNA) but no the infectious gene. The shRNA gene transfused into lung cancer cells will recognize and complement the specific target mRNA sites of the CYP1A1 and YWHAZ genes. After that, shRNA will cut the mRNA which should be expressed into a cancer protein. Unexpressed mRNA into protein causes cancer cell growth inhibited. Thus, this gene therapy solution is expected to be able to reduce the rate of death caused by lung cancer. Keywords: Lung cancer, CYP1A1 and YWHAZ genes, gene therapy, lentiviral, shRNA

Transcription expression of gene encoding cathelicidin &lt;i&gt; CATHL4&lt;/i&gt; in Vietnam indigenous yellow cattle

Cathelicidins, a family of host-defence peptides, are present in a diverse range of species, including fish, amphibians, birds, reptiles, and mammals. The evidence for the role of these cationic antimicrobial peptides in innate host defenses is convincing from the data of animal model and transgenic animal experiments as well as wildlife or domestic animals, indicating that the peptides protect against inflammatory and immune system after induction by bacterial infection. In this study, we present the assessment of transcription expression of CATHL4 gene which encodes indolicidin, a cathelicidin from indigenous yellow cattle of Vietnam. The research focused on RNA samples extracted from lung tissues and lymph node collected from diseased cattle which died of infectious respiratory symptoms and the healthy cattle which were slaughtered for food purposes. By quantitative real-time PCR, the relative expression of transcripts was determined and analyzed using the expression of YWHAZ gene as reference. The results showed that this gene was abundantly expressed at a higher level in tissues of the diseased cattle than in those of the healthy ones. In both infection and healthy states, the expression of CATHL4 in lymph nodes were higher than in lung tissues. This indicated that CATHL4 (indolicidin) may participate in functions against infectious pathogens.

The exploration of a new environment leads to the modulation of gene expression for prolonged times in the rat.

In the present study we performed a transcriptional analysis in order to evaluate changes in gene expression induced by exploration in prolonged times. The analysis was carried out 3, 10 and 20 days after exploration. We analyzed the modulation of the expression levels of Pfn2, Casp3, Pdrg1, Pea15, Ywhaz genes which previously were found not modulated 2 days after exploration. Our data show that the expression of Pfn2, Casp3, Pdrg1, Pea15, Ywhaz genes was modulated at 10 or 20 days. The transcript, whose expression had been evaluated with the qRT-PCR, code for proteins which belong to the following functional categories: synaptic modulation, apoptosis, signal transduction. It is interesting to note that the modulation of the expression of these genes was evident some days after environmental exploration, and not previously at 2 days after conditioning as occurred after contextual fear conditioning (CFC). Hence it is possible to hypothesize that the spatial memory processes require a longer period of elaboration than the emotional ones, fundamental for the survival of the species.

Dynamic transcriptome analysis unveils key proresolving factors of chronic inflammatory arthritis.

Despite recent advances in understanding chronic inflammation remission, global analyses have not been explored to systematically discover genes or pathways underlying the resolution dynamics of chronic inflammatory diseases. Here, we performed time-course gene expression profiling of mouse synovial tissues along progression and resolution of collagen-induced arthritis (CIA) and identified genes associated with inflammation resolution. Through network analysis of these genes, we predicted 3 key secretory factors responsible for the resolution of CIA: Itgb1, Rps3, and Ywhaz. These factors were predominantly expressed by Tregs and antiinflammatory M2 macrophages, suppressing production of proinflammatory cytokines. In particular, Ywhaz was elevated in the sera of mice with arthritis resolution and in the urine of rheumatoid arthritis (RA) patients with good therapeutic responses. Moreover, adenovirus-mediated transfer of the Ywhaz gene to the affected joints substantially inhibited arthritis progression in mice with CIA and suppressed expression of proinflammatory cytokines in joint tissues, lymph nodes, and spleens, suggesting Ywhaz is an excellent target for RA therapy. Therefore, our comprehensive analysis of dynamic synovial transcriptomes provides previously unidentified antiarthritic genes, Itgb1, Rps3, and Ywhaz, which can serve as molecular markers to predict disease remission, as well as therapeutic targets for chronic inflammatory arthritis.

PPIA, HPRT1, and YWHAZ Genes Are Suitable for Normalization of mRNA Expression in Long-Term Expanded Human Mesenchymal Stem Cells.

Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.

YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance by suppressing caspase-mediated apoptosis.

The objective of this study was to characterize the oncogenic actions of a recently identified cancer‐associated gene YWHAZ (also named as 14‐3‐3 ζ/δ) in urothelial carcinomas of the urinary bladder (UCUB). A genome‐wide study revealed YWHAZ to be involved in the amplicon at 8q22.3, and its genetic amplification was detected predominantly in muscle‐invasive bladder cancer (MIBC). Immunohistochemical staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis and STRING network studies suggested involvement of YWHAZ in regulating caspase‐mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low endogenous YWHAZ levels boosted cell resistance to doxorubicin and cisplatin, as well as to ionizing radiation. Conversely, YWHAZ‐knockdown using specific shRNA in cells with high endogenous YWHAZ levels diminished survival activity, suppressing cell growth and increasing cell death. Our findings confirm the essential role played by YWHAZ in sustaining cell proliferation during chemo/radiotherapy. Treatments based on anti‐YWHAZ strategies may thus be beneficial for UCUB patients overexpressing YWHAZ. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

MiR-375-3p/YWHAZ/β-catenin axis regulates migration, invasion, EMT in gastric cancer cells.

YWHAZ (14-3-3ζ) plays crucial roles in regulating proliferation, apoptosis, migration, and invasion of gastric cancer (GC) cells. However, its extensive roles and potential mechanisms in GC cells remain unknown, and need to be researched deeply. In this study, we focus on the role of miR-375/YWHAZ axis in migration, invasion and epithelial-to-mesenchymal transition (EMT) of GC cells. YWHAZ level was assessed by western blot and qPCR assays in GC cells. Scratch and transwell assays were used to determine the migration and invasion of GC cells. The protein levels of correlative molecules were detected by western blot. The regulation of miR-375 on the expression of its target gene YWHAZ was verified by dual-luciferase report system. According to the results, knockdown of YWHAZ inhibited the migration, invasion and EMT of GC cells. Moreover, silencing of YWHAZ restrained the activation of wnt/β-catenin signalling pathway. YWHAZ was confirmed to be a target gene of miR-375, and its expression was regulated by miR-375 in GC cells. Transfection of miR-375 inhibitor promoted the migration, invasion, EMT and activation of wnt/β-catenin pathway in GC cells, which was suppressed by inhibition of YWHAZ. Taken together, this study suggests that miR-375/YWHAZ axis may be served as a novel therapeutic target for GC patients.

Knockdown of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) enhances tumorigenesis both in vivo and in vitro in bladder cancer.

Bladder cancer is the most common tumor of the urinary tract. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, has been observed to be frequently amplified in bladder cancer. However, the role of 14-3-3ζ in various types of cancer is controversial. With reproduction of The Cancer Genome Atlas database, we searched the correlation of YWHAZ expression with survival outcomes of multiple cancers in silico. Our results revealed that only in bladder cancer was there a positive survival trend with YWHAZ overexpression. To study its role in bladder cancer, YWHAZ was successfully downregulated by lentivirus in 5637 and T24 cells. MTT and colony-formation assays showed that YWHAZ downregulation increased cell proliferation. Wound healing and Transwell assays showed that YWHAZ downregulation enhanced cell migration and invasiveness. FACS analysis showed that YWHAZ induced cell cycle arrest, but not apoptosis. A xenograft tumor model revealed that YWHAZ knockdown enhanced tumor growth. Gene set enrichment analysis confirmed that the cell cycle pathway plays a vital role in the function of the YWHAZ gene. In conclusion, knockdown of YWHAZ promoted both in vitro and in vivo tumorigenesis in bladder cancer and may be a novel biomarker for bladder cancer deserving further study.

M13 - Contribution of The 14-3-3 Gene Family To Autism Spectrum Disorder

Background We recently identified a heterozygous 1-bp insertion in the YWHAZ gene (c.659_660insT, p.L220Ffs*18) in two brothers with Autism Spectrum Disorder (ASD) (Toma et al. Mol Psychiatry 2014;19:784). This gene encodes 14-3-3ζ, one of the seven isoforms of the 14-3-3 protein family. In this study we aimed to (1) functionally characterize the disruptive mutation identified in YWHAZ; and (2) evaluate the possible implication in ASD of all members of this gene family (SFN, YWHAQ, YWHAG, YWHAZ, YWHAB, YWHAH, YWHAE). Methods Normal and truncated 14-3-3ζ were expressed as fusion proteins in E. coli, where their solubility was tested. Furthermore, Biacore experiments were performed to assess the affinity of the truncated 14-3-3ζ with the well-known interactor Ser19-phosphorylated tyrosine hydroxylase (TH-Ser19P). To test the possible involvement of common and rare variants of the 14-3-3 genes in ASD we performed, respectively, (a) a case-control association study in 727 ASD patients and 714 controls, by using tagSNPs covering the seven genes; and (b) resequencing of all these genes in 285 ASD patients. Results Truncated 14-3-3ζ presented a decreased solubility and lost its affinity with TH-Ser19P. No common variants were found associated with ASD in our study. The mutation screening identified two potentially damaging variants in the SFN gene. Discussion These results suggest a possible dominant negative effect of the truncated 14-3-3ζ. The genetic study of the 14-3-3 gene family did not identify other risk variants in the YWHAZ gene. Although potential mutations were identified in SFN, further experiments are needed to test its possible contribution to ASD.

Whole-genome expression analysis reveals genes associated with treatment response to escitalopram in major depression

The reasons for variability in treatment response in major depressive disorder (MDD) are not fully understood, but there is accumulating evidence suggesting that therapeutic outcomes of antidepressants can be influenced by genetic factors. In the present study we applied the microarray Illumina platform for whole genome expression profiling in depressive patients treated with escitalopram medication in order to identify genes underlying response to antidepressant treatment. The initial study sample consisted of 135 outpatients with major depressive disorder (mean age 31.1±11.6 years, 68% females) treated with escitalopram 10–20mg/day for 12 weeks, from which 87 patients (55 females) were included in gene expression analyzing. The gene expression profiles were measured on peripheral blood cells at baseline, at week 4 and at the end of treatment (week 12) using BeadChips Illumina. The fold change was used to demonstrate rate of changes in average gene expressions between studied groups. Statistical analyses were performed using the false discovery rate (FDR). The most interesting gene, which showed the predictive effect on treatment outcome by delineating low dose responders and treatment-resistant patients at the beginning of medication, was NLGN2, belonging to a family of neuronal cell surface proteins and involving in synapse formation. In addition, the several gene clusters, related to immune response, signal transduction and neurotrophin pathway, have distinguished responders from non-responders at the week 4 of treatment. After 4 weeks of escitalopram treatment (10mg/day), the YWHAZ gene has showed the highest transcriptional change in responders as compared with non-responders. Finally, at the end of the treatment we noticed that at least three genes (NR2C2, ZNF641, FKBP1A) have been strongly associated with resistance to escitalopram. Thus the results of this study support that exploration of peripheral gene expression is a useful tool in the further identification of novel genetic biomarkers for antidepressant treatment response.