Mowat-Wilson syndrome (MWS) is a rare autosomal dominant genetic disease caused by zinc finger E-box-binding homeobox 2 (ZEB2) gene mutation and has various clinical manifestations including intellectual disability/global developmental delay, unusual facies and multiple congenital malformations. This article reports the clinical features and gene mutations of three children diagnosed with MWS by ZEB2 gene analysis. All three children had Hirschsprung disease and unusual facies. One child died of severe heart failure and pneumonia at the age of 4 months. Global developmental delay was not discovered by her parents due to her young age. The other two children had severe global developmental delay. All three children carried a de novo heterozygous nonsense mutation in the ZEB2 gene, among which c.756C>A (p.Y252X) had not been reported before. Such mutations produced truncated proteins and were highly pathogenic. MWS is presented with strong clinical and genetic heterogeneity. Clinicians should consider the possibility of MWS when a child has unusual facies of MWS, intellectual disability/global developmental delay and multiple congenital malformations. Gene detection helps to make a confirmed diagnosis.

Differences in fast beta (20-28Hz) electroencephalogram (EEG) oscillatory activity distinguish some individuals with psychiatric and substance use disorders, suggesting that it may be a useful endophenotype for studying the genetics of disorders characterized by neural hyper-excitability. Despite the high heritability estimates provided by twin and family studies, there have been relatively few genetic studies of beta EEG, and to date only one genetic association finding has replicated (i.e., GABRA2).

In a sample of 1564 individuals from 117 families of European Ancestry (EA) drawn from the Collaborative Study on the Genetics of Alcoholism (COGA), we performed a Genome-Wide Association Study (GWAS) on resting-state fronto-central fast beta EEG power, adjusting regression models for family relatedness, age, sex, and ancestry. To further characterize genetic findings, we examined the functional and behavioral significance of GWAS findings.

Three intronic variants located within DSE (dermatan sulfate epimerase) on 6q22 were associated with fast beta EEG at a genome wide significant level (p<5×10-8). The most significant SNP was rs2252790 (p<2.6×10-8; MAF=0.36; β=0.135). rs2252790 is an eQTL for ROS1 expressed most robustly in the temporal cortex (p=1.2×10-6) and for DSE/TSPYL4 expressed most robustly in the hippocampus (p=7.3×10-4; β=0.29). Previous studies have indicated that DSE is involved in a network of genes integral to membrane organization; gene-based tests indicated that several variants within this network (i.e., DSE, ZEB2, RND3, MCTP1, and CTBP2) were also associated with beta EEG (empirical p<0.05), and of these genes, ZEB2 and CTBP2 were associated with DSM-V Alcohol Use Disorder (AUD; empirical p<0.05).'

In this sample of EA families enriched for AUDs, fast beta EEG is associated with variants within DSE on 6q22; the most significant SNP influences the mRNA expression of DSE and ROS1 in hippocampus and temporal cortex, brain regions important for beta EEG activity. Gene-based tests suggest evidence of association with related genes, ZEB2, RND3, MCTP1, CTBP2, and beta EEG. Converging data from GWAS, gene expression, and gene-networks presented in this study provide support for the role of genetic variants within DSE and related genes in neural hyperexcitability, and has highlighted two potential candidate genes for AUD and/or related neurological conditions: ZEB2 and CTBP2. However, results must be replicated in large, independent samples.

Objective: To investigate the effect of zinc finger E-box-binding homeobox 2 (ZEB2) on hepatitis B virus (HBV) replication and expression. Methods: HepG2, HepG2.2.15, and HepAD38 cells were cultured separately, and Western blot was used to measure the expression of ZEB2. HepG2.2.15 cells were cultured and transfected with ZEB2 expression plasmids or shRNA targeting ZEB2. Western blot was used to measure the expression of ZEB2 and HBV core proteins, quantitative real-time PCR was used to measure HBV 3.5 kb RNA and HBV DNA, Southern blot was used to measure HBV replicative intermediate, and ELISA was used to measure the expression of HBsAg and HBeAg, in order to clarify the effect of ZEB2 on HBV replication and expression. The dual-luciferase reporter system was used to analyze the effect of ZEB2 on HBV promoter, and the chromatin immunoprecipitation assay was used to detect the binding of ZEB2 to HBV promoter. The t-test was used for comparison of means between groups. Results: The expression of ZEB2 was inhibited in the cells with HBV replication. Overexpression of ZEB2 reduced the level of HBV replication and expression by about 50% (P< 0.05). After ZEB2 was downregulated by shZEB2-1 or shZEB2-2, the level of HBV replicative intermediate increased from 58.53 ± 3.43 to 112.80 ± 5.03, and 128.30 ± 2.31, the relative expression level of HBV 3.5 kb RNA increased from 1.00 ± 0.01 to 2.03 ± 0.02 and 2.32 ± 0.03, the level of HBsAg increased from 35.63% ± 1.57% to 81.87% ± 0.43% and 100.00% ± 2.18%, and HBeAg increased from 37.00% ± 0.70% to 88.00% ± 2.60% and 100.00% ± 0.75%. Furthermore, ZEB2 could bind to HBV core promoter and inhibit its transcriptional activity. Conclusion: ZEB2 inhibits HBV replication and expression through binding to HBV core promoter and inhibiting its transcriptional activity.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect. The etiology of NSCLP is complex with multiple genes and environmental factors playing causal roles. Although studies have identified numerous genetic markers associated with NSCLP, the role of epigenetic variation remains relatively unexplored. Because of their identical DNA sequences, monozygotic (MZ) twins discordant for NSCLP are an ideal model for examining the potential contribution of DNA methylation to non-syndromic orofacial clefting. In this study, we compared the patterns of whole genome DNA methylation in six MZ twin pairs discordant for NSCLP. Differentially methylated positions (DMPs) and regions (DMRs) were identified in NSCLP candidate genes, including differential methylation in MAFB and ZEB2 in two independent MZ twin pairs. In addition to DNA methylation differences in NSCLP candidate genes, we found common differential methylation in genes belonging to the Hippo signaling pathway, implicating this mechanosensory pathway in the etiology of NSCLP. The results of this novel approach using MZ twins discordant for NSCLP suggests that differential methylation is one mechanism contributing to NSCLP, meriting future studies on the role of DNA methylation in familial and sporadic NSCLP.

Keywords: NSCLP; methylation; non-syndromic cleft lip and cleft palate; twins; whole genome bisulfite sequencing.

Objective: β-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We have previously shown that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes.

Method: We performed bulk and single-cell RNA-Sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-βH1 cell lines. We analysed previously published mouse and human T2D datasets.

Results: Βulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial to mesenchymal transition (EMT) in pre-diabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell:β-cell contacts, increased extracellular matrix (ECM) deposition and TGFβ-dependent islet fibrosis. We find that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor ZEB2. Following compromised β-cell identity, the reduction of Pdx1 gene expression causes a decrease of Ovol2 protein, thereby triggering mesenchymal reprogramming of β-cells through the induction of Zeb2. Finally, we provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islet of T2D subjects.

Conclusions: Our study indicates that miR-7-mediated β-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predispose to fibrosis. This work suggests that regulators of EMT signalling may represent novel therapeutic targets for the treatment of β-cell dysfunction and fibrosis in T2D.

Keywords: dedifferentiation; diabetes; epithelial-to-mesenchymal transition; insulin; microRNA; pancreatic β-cells.

How does the immune system tailor effector function to particular threats? Krueger et al. reveal that infection with Salmonella enterica (SE), but not with influenza A virus (IAV), drives interleukin (IL)-12-dependent outgrowth of interferon (IFN)-γ^hi type 1 T helper (Th1) cells, leading to superior protection against this phagosomal pathogen. Among these cells are ZEB2-dependent cytotoxic Th1 cells marked by CX3CR1 expression.

Keywords: CX3CR1; IL-12; T helper cells; ZEB2; cytotoxic CD4 T cells; phagosomal pathogens.

[This corrects the article DOI: 10.1371/journal.pone.0209497.].

Gastric cancer is a malignancy with high incidence and mortality worldwide. In gastric cancer, epithelial-mesenchymal transition (EMT) and metastasis further increase the mortality rate. Trefoil factor 1 (TFF1) has been reported as a protective factor in the gastric mucosa. In this study, TFF1 inhibited the migration and invasive capability of gastric cancer cells. Elevated TFF1 levels induced the expression of E-cadherin, the epithelial marker, and reduced the expression of N-cadherin, vimentin, Snail, Twist, Zinc finger E-box binding homeobox (ZEB) 1 and ZEB2, well-known repressors of E-cadherin expression. In addition, the expression of matrix metalloproteinase (MMP)-2, MMP-7 and MMP-9, which are major markers of cancer metastasis, was suppressed by TFF1. Upregulation of TFF1 inhibited TGF-β, a major signaling for EMT induction, and the phosphorylation of Smad2/3 activated by TGF-β in AGS cells. In conclusion, TFF1 inhibits EMT through suppression of TGF-β signaling in AGS cells, which might be used in therapeutic strategies for reducing metastatic potential and invasiveness of these cells.

Keywords: Epithelial-mesenchymal transition; Gastric cancer; Transforming growth factor beta; Trefoil factor 1.

Colon cancer is a malignant tumor that seriously affects human health. Recently, studies revealed that the expression of MTBP enhanced the proliferation and metastasis of many types of cancer cells. And the data also showed that MTBP has the potential to regulate the expression of ZEB2. However, it is unclear whether MTBP can affect the proliferation, migration and invasion of colon cancer cells by modulating the expression of ZEB2. In this study, we established the MTBP overexpression and knockdown colon cancer cells with the transfection. Next, CCK-8 and transwell assays were carried out to determine the changes of the proliferation and invasion of colon cancer cells, respectively. After that, we overexpressed the ZEB2 in these MTBP knockdown colon cancer cells. Finally, the invasion and migration of these cells were detected with the same methods. We revealed that overexpression of MTBP enhanced the proliferation and invasion of colon cancer cells. Moreover, suppression of MTBP repressed the proliferation, migration and invasion of colon cancer cells. Furthermore, MTBP promoted the expression of ZEB2. The overexpression of ZEB2 abolished the MTBP knockdown induced inhibition of the migration and invasion of colon cancer cells. These results implied that MTBP enhanced the proliferation, migration and invasion of colon cancer cells by activating the expression of ZEB2.

Keywords: Colon cancer cells; MTBP; ZEB2; invasion; proliferation.

Purpose: The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated.

Methods: In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17β-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response.

Results: Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17β-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells.

Conclusion: ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.

Keywords: Breast cancer; Endocrine therapy resistance; Estrogen; Gene regulation; Luminal A; Metastasis; ZEB2.

Background: Diesel exhaust particles (DEP) are associated with the prevalence and exacerbation of allergic respiratory diseases, including allergic rhinitis and allergic asthma. However, DEP-induced mechanistic pathways promoting upper airway disease and their clinical implications remain unclear OBJECTIVE: We sought to investigate the mechanisms by which DEP exposure contributes to nasal polyposis using human-derived epithelial cells and a murine nasal polyp (NP) model.

Methods: Gene set enrichment and weighted gene co-expression network analyses were performed. Cytotoxicity, epithelial-to-mesenchymal transition (EMT) markers, and nasal polyposis were assessed. Effects of DEP exposure on EMT were determined using epithelial cells from normal or CRS patients with or without NPs. BALB/c mice were exposed to DEP through either a nose-only exposure system or nasal instillation, with or without house dust mite (HDM), followed by ZEB2 shRNA delivery.

Results: Bioinformatics analyses revealed that DEP exposure triggered EMT features in airway epithelial cells (AECs). Similarly, DEP-exposed human nasal epithelial cells (hNECs) exhibited EMT characteristics, which were dependent on ZEB2 expression. hNEC-derived from chronic rhinosinusitis (CRS) patients presented more prominent EMT features after DEP treatment, when compared to those from control subjects and NP patients. Co-exposure to DEP and HDM synergistically increased the number of NPs, epithelial disruptions, and ZEB2 expression. Most importantly, ZEB2 inhibition prevented DEP-induced EMT, thereby alleviating NP formation in mice.

Conclusions: Our data show that DEP facilitated NP formation, possibly via the promotion of ZEB2-induced EMT. ZEB2 may be a therapeutic target for DEP-induced epithelial damage and related airway diseases, including NPs.

Keywords: Diesel exhaust particles; ZEB2; epithelial-to-mesenchymal transition; nasal polyps; upper airway inflammatory diseases.

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteosome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production.

Glioblastomas (GBMs) are the most frequent primary malignancies in the central nervous system. Aberrant activation of WNT/β-catenin signaling pathways is critical for GBM malignancy. However, the regulation of WNT/β-catenin signaling cascades remains unclear. Presently, we observed the increased expression of ZEB2 and the decreased expression of miR-637 in GBM. The expression of miR-637 was negatively correlated with ZEB2 expression. miR-637 overexpression overcame the ZEB2-enhanced cell proliferation and G1/S phase transition. Besides, miR-637 suppressed the canonical WNT/β-catenin pathways by targeting WNT7A directly. Gain- and loss-of-function experiments with U251 mice demonstrated that miR-637 inhibited cell proliferation and arrested the G1/S phase transition, leading to tumor growth suppression. The collective findings suggest that ZEB2 and WNT/β-catenin cascades merge at miR-637, and the ectopic expression of miR-637 disturbs ZEB2/WNT/β-catenin-mediated GBM growth. The findings provide new clues for improving β-catenin-targeted therapy against GBM.

Keywords: Glioblastoma progression; WNT7A; ZEB2; miR-637; β-catenin.

There is emerging evidence suggesting that epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) play an important role in colorectal carcinoma (CRC), but their exact role remains controversial. Our aim was to analyze the miR-200 family as EMT markers and their target genes expression at invasive tumor front and in nodal and liver metastases. Sixty-three formalin-fixed paraffin-embedded tissue samples from 19 patients with CRC were included. Using a micropuncture technique, tissue was obtained from central part and invasive front of the primary tumor, and nodal and liver metastases. Expression of the miR-200 family and their target genes CDKN1B, ONECUT2, PTPN13, RND3, SOX2, TGFB2 and ZEB2 was analyzed using real-time PCR. We found miR-200 family down-regulation at invasive front compared to central part, and up-regulation of miRNA-200a/b/c and miR-429 in metastases compared to invasive front. At invasive front, TGFB2 was the only gene with inverse expression to the miR-200 family, whereas in metastases inverse expression was found for ONECUT2 and SOX2. CDKN1B, PTPN13 and ZEB2 were down-regulated at invasive front and up-regulated in metastases. Our results suggest the involvement of partial EMT at invasive tumor front, and partial MET in metastases in CRC, based on miR-200 family and its target genes expression.

Keywords: colon cancer; epithelial-mesenchymal transition; mesenchymal-epithelial transition; metastases; miRNA.

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease globally. miRNAs (miRs) regulate various cellular events that lead to NAFLD. In this study we tested the hypothesis that miR-155 is an important regulator of steatohepatitis and fibrosis pathways. Wild type (WT) or miR-155 deficient (KO) mice received a high fat-high cholesterol-high sugar-diet (HF-HC-HS) for 34 weeks and liver tissues were analyzed. In patients with nonalcoholic steatohepatitis and in the mouse model of HF-HC-HS diet we found increased miR-155 levels in the liver compared to normal livers. Upon HF-HC-HS diet feeding, miR-155 KO mice displayed less liver injury, decreased steatosis, and attenuation in fibrosis compared to WT mice. ALT, triglyceride levels, and genes involved in fatty acid metabolic pathway were increased in WT mice whereas miR-155 KO mice showed attenuation in these parameters. HF-HC-HS diet-induced significant increase in the expression of NLRP3 inflammasome components in the livers of WT mice compared to chow fed diet. Compared to WT mice, miR-155 KO showed attenuated induction in the NLRP3, ASC, and caspase1 inflammasome expression on HF-HC-HS diet. Fibrosis markers such as collagen content and deposition, αSMA, Zeb2, and vimentin were all increased in WT mice and miR-155 KO mice showed attenuated fibrosis marker expression. Overall, our findings highlight a role for miR-155 in HF-HC-HS diet-induced steatosis and liver fibrosis.

Purpose: Mowat-Wilson syndrome (MWS) is a rare complex malformation syndrome which is characterized by typical facial dysmorphism, moderate to severe intellectual disability, global developmental delay, and multiple congenital anomalies. Here, we summarize the clinical characteristics and gene mutation analysis of a Chinese boy with MWS.

Patients and methods: The clinical features of the patient were monitored. DNA extracted from peripheral blood was subjected to sequencing analysis. Then, the whole-exome sequencing was performed.

Results: A novel deletion mutation (c.1137_1146del TAGTATGTCT) was identified in exon 8 of the ZEB2 gene. The deletion mutation was predicted to produce a truncated protein (p.S380Nfs*13), resulting in haploinsufficiency. The patient presented with short stature, microcephaly, congenital heart defects, cryptorchidism, corpus callosum agenesis, global developmental delay, and intellectual disability. Furthermore, he demonstrated bilateral sensorineural hearing loss. This manifestation is less common in MWS. It is first reported in Chinese patients with MWS. Clinical follow-up showed that the facial features of MWS developed with time. The facial features of the patient were not obvious except for the uplifted ear lobes at the age of 3 months. At the age of 22 months, the facial characteristics of the patient included ocular hypertelorism, frontal bossing, rounded nasal tip, sparse eyebrows, prominent chin, widely spaced teeth, and uplifted ear lobes with a central depression.

Conclusion: A novel deletion mutation of the ZEB2 gene was identified. This work contributes to expanding the mutation spectra of MWS. Our results may reflect the variability of the phenotype in MWS.

Keywords: Mowat–Wilson syndrome; ZEB2; gene mutation; phenotype.

Background and aims: Coronary artery disease (CAD) arises from the interaction of genetic and environmental factors. Although genome-wide association studies (GWAS) have identified multiple risk loci and single nucleotide polymorphisms (SNPs) associated with risk of CAD, they are predominantly located in non-coding or intergenic regions and their mechanisms of effect are largely unknown. Accordingly, our objective was to develop a data-driven informatics pipeline to understand complex CAD risk loci, and to apply this to a poorly understood cluster of SNPs in the vicinity of ZEB2.

Methods: We developed a unique informatics pipeline leveraging a multi-tissue CAD genetics-of-gene-expression dataset, GWAS datasets, and other resources. The pipeline first dissected SNP locations and their linkage disequilibrium relationships, and progressed through analyses of tissue-specific expression quantitative trait loci, and then gene-gene, gene-phenotype, SNP-phenotype relationships. The pipeline concluded by exploring CAD-relevant gene regulatory networks (GRNs).

Results: We identified three independent CAD risk SNPs in close proximity to the ZEB2 coding region (rs6740731, rs17678683 and rs2252641/rs1830321). Our pipeline determined that these SNPs likely act in concert via the atherosclerotic arterial wall and adipose tissues, by governing metabolic and lipid functions. In addition, ZEB2 is the top key driver of a liver-specific GRN that is related to lipid levels, metabolic and anthropometric measures, and CAD severity.

Conclusions: Using a novel informatics pipeline, we disclosed the multi-faceted mechanisms of action of the ZEB2-associated CAD risk SNPs. This pipeline can serve as a roadmap to dissect complex SNP-gene-tissue-phenotype relationships and to reveal targets for tissue- and gene-specific therapeutic interventions.

Keywords: Atherosclerosis; Coronary artery disease; Genome-wide association study; ZEB2.

Introduction: Acute myeloid leukemia (AML) is the most prevalent type of adult hematopoietic system leukemia. Conventional therapies are associated with unfavorable side effects in individuals diagnosed with AML. These aftereffects with partial remission reflect the urgent need for novel therapeutic approaches for inducing apoptosis, specifically in malignant cells without affecting other cells. As a transcription factor (TF), ZEB2 (Zinc Finger E-Box Binding Homeobox 2), regulates the expression of specific genes in normal conditions. However, increased expression of ZEB2 is reported in various cancers, especially in AML, which is related to a higher degree of apoptosis inhibition of malignant cells. In this work, the role of ZEB2 in apoptosis inhibition is surveyed through ZEB2 specific knocking-down in human myeloid leukemia HL-60 cells.

Materials and methods: Transfection of HL-60 cells was conducted using ZEB2-siRNA at concentrations of 20, 40, 60, and 80 pmol within 24, 48, and 72 h. After determining the optimum dose and time, flow cytometry was used to measure the apoptosis rate. The MTT assay was also applied to evaluate the cytotoxic impact of transfection on the cells. The expression of candidate genes was measured before and after transfection using qRT-PCR.

Results: According to obtained results, suppression of ZEB2 expression through siRNA was associated with the induction of apoptosis, increased pro-apoptotic, and decreased anti-apoptotic gene expression. Transfection of ZEB2-siRNA was also associated with reduced cell proliferation and viability.

Conclusion: Our study results suggest that ZEB2 suppression in myeloid leukemia cells through apoptosis induction could be a proper therapeutic method.

Keywords: Acute myeloid leukemia (AML); Apoptosis.; RNAi; Targeted therapy; ZEB2 (Zinc Finger E-Box Binding Homeobox 2).

Zinc finger E-box-binding homeobox 2 (ZEB2) has been reported to mediate epithelial-mesenchymal transition (EMT) and disease progression in several cancer types. However, the expression of ZEB2 in esophageal squamous cell carcinoma (OSCC) and its association with prognosis remains unclear. In the present study, a tissue microarray and immunohistochemistry were used to investigate ZEB2 and epithelial (E-)cadherin expression in OSCC tissues (n=218) and peritumoral esophageal tissues (POT; n=60). There was a significantly increased incidence of positive ZEB2 expression in OSCC tissues compared with the expression in POTs (P<0.012). By contrast, the incidence of positive E-cadherin expression in OSCC tissues was significantly decreased compared with the expression in POTs (P<0.004). ZEB2 expression in OSCC was associated with a number of clinicopathological factors, and it was also an independent predictive factor for shorter overall survival time (P<0.001). Overall, ZEB2 may promote OSCC metastasis and is a potential prognostic marker for malignancy.

Objective: This study aims to explore whether ZEB2-AS1 can promote the development of osteosarcoma by affecting the proliferation, invasion, and apoptosis of osteosarcoma cells.

Patients and methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the ZEB2-AS1 expression in osteosarcoma tissue specimens and normal bone tissues. After ZEB2-AS1 downregulation, Cell Counting Kit-8 (CCK-8) test, plate cloning assay, 5-Ethynyl-2'-deoxyuridine (EdU) experiment, and flow cytometry were conducted to analyze the changes in cell proliferation and apoptosis. RIP assay was performed to detect the binding of ZEB2-AS1 to EZH2, while Western blot was applied to examine the EZH2 expression after EZH2 was inhibited. Meanwhile, after simultaneously inhibiting ZEB2-AS1 and EZH2, the cell invasiveness was determined by transwell assay.

Results: ZEB2-AS1 was highly expressed in osteosarcoma tissues, especially in advanced and metastatic groups. Interfering with ZEB2-AS1 suppressed cell proliferation and enhanced cell apoptosis. In addition, ZEB2-AS1 was confirmed to be able to combine with EZH2. The knockdown of ZEB2-AS1 attenuated the cell invasion ability, which was further decreased after the simultaneous downregulation of ZEB2-AS1 and EZH2.

Conclusions: The long non-coding RNA, ZEB2-AS1, enhanced the proliferation and invasion of osteosarcoma cells and inhibited the cell apoptosis by combining with EZH2, and thereby promoted the development of osteosarcoma.