Zinc Finger E-box Binding Homeobox Research Articles

Differential Expression of SRY-Related HMG-Box Transcription Factor 2, Oligodendrocyte Lineage Transcription Factor 2, and Zinc Finger E-Box Binding Homeobox 1 in Serum-Derived Extracellular Vesicles: Implications for Mithramycin Sensitivity and Targeted Therapy in High-Grade Glioma.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and is often resistant to traditional therapies. Evidence suggests that glioma stem cells (GSCs) contribute to this resistance. Mithramycin (Mit-A) targets GSCs and exhibits antitumor activity in GBM by affecting transcriptional targets such as SRY-related HMG-box transcription factor 2 (SOX2), oligodendrocyte lineage transcription factor 2 (OLIG2), and zinc finger E-box binding homeobox 1 (ZEB1). However, its clinical use has been limited by toxicity. This study explored the diagnostic potential of serum extracellular vesicles (EVs) to identify Mit-A responders. Serum EVs were isolated from 70 glioma patients, and targeted gene expression was analyzed using qRT-PCR. Using chemosensitivity assay, we identified 8 Mit-A responders and 17 nonresponders among 25 glioma patients. The M-score showed a significant correlation (p = 0.045) with isocitrate dehydrogenase 1 mutation but not other clinical variables. The genes SOX2 (p = 0.005), OLIG2 (p = 0.003), and ZEB1 (p = 0.0281) were found to be upregulated in the responder EVs. SOX2 had the highest diagnostic potential (AUC = 0.875), followed by OLIG2 (AUC = 0.772) and ZEB1 (AUC = 0.632).The combined gene panel showed significant diagnostic efficacy (AUC = 0.956) through logistic regression analysis. The gene panel was further validated in the serum EVs of 45 glioma patients. These findings highlight the potential of Mit-A as a targeted therapy for high-grade glioma based on differential gene expression in serum EVs. The gene panel could serve as a diagnostic tool to predict Mit-A sensitivity, offering a promising approach for personalized treatment strategies and emphasizing the role of GSCs in therapeutic resistance.

LncRNA PSMB8-AS1 Instigates Vascular Inflammation to Aggravate Atherosclerosis.

Increasing evidence suggests that long noncoding RNAs play significant roles in vascular biology and disease development. One such long noncoding RNA, PSMB8-AS1, has been implicated in the development of tumors. Nevertheless, the precise role of PSMB8-AS1 in cardiovascular diseases, particularly atherosclerosis, has not been thoroughly elucidated. Thus, the primary aim of this investigation is to assess the influence of PSMB8-AS1 on vascular inflammation and the initiation of atherosclerosis. We generated PSMB8-AS1 knockin and Apoe (Apolipoprotein E) knockout mice (Apoe-/-PSMB8-AS1KI) and global Apoe and proteasome subunit-β type-9 (Psmb9) double knockout mice (Apoe-/-Psmb9-/-). To explore the roles of PSMB8-AS1 and Psmb9 in atherosclerosis, we fed the mice with a Western diet for 12 weeks. Long noncoding RNA PSMB8-AS1 is significantly elevated in human atherosclerotic plaques. Strikingly, Apoe-/-PSMB8-AS1KI mice exhibited increased atherosclerosis development, plaque vulnerability, and vascular inflammation compared with Apoe-/- mice. Moreover, the levels of VCAM1 (vascular adhesion molecule 1) and ICAM1 (intracellular adhesion molecule 1) were significantly upregulated in atherosclerotic lesions and serum of Apoe-/-PSMB8-AS1KI mice. Consistently, in vitro gain- and loss-of-function studies demonstrated that PSMB8-AS1 induced monocyte/macrophage adhesion to endothelial cells and increased VCAM1 and ICAM1 levels in a PSMB9-dependent manner. Mechanistic studies revealed that PSMB8-AS1 induced PSMB9 transcription by recruiting the transcription factor NONO (non-POU domain-containing octamer-binding protein) and binding to the PSMB9 promoter. PSMB9 (proteasome subunit-β type-9) elevated VCAM1 and ICAM1 expression via the upregulation of ZEB1 (zinc finger E-box-binding homeobox 1). Psmb9 deficiency decreased atherosclerotic lesion size, plaque vulnerability, and vascular inflammation in Apoe-/- mice in vivo. Importantly, endothelial overexpression of PSMB8-AS1-increased atherosclerosis and vascular inflammation were attenuated by Psmb9 knockout. PSMB8-AS1 promotes vascular inflammation and atherosclerosis via the NONO/PSMB9/ZEB1 axis. Our findings support the development of new long noncoding RNA-based strategies to counteract atherosclerotic cardiovascular disease.

Astragaloside IV inhibits epithelial-mesenchymal transition and pulmonary fibrosis via lncRNA-ATB/miR-200c/ZEB1 signaling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease involving multiple factors and genes. Astragaloside IV (ASV) is one of the main bioactive ingredients extracted from the root of Astragalus membranaceus, which plays an important role in anti-inflammatory, antioxidant and improve cardiopulmonary function. Epithelial-mesenchymal transition (EMT) is a key driver of the process of pulmonary fibrosis, and Zinc finger E-box-binding homeobox 1 (ZEB1) can promote pulmonary fibrosis in an EMT-dependent manner. Here, we found that ASV effectively inhibited the ZEB1 and EMT in both bleomycin (BLM)-induced rat pulmonary fibrosis and TGF-β1-treated A549 cells. To further elucidate the molecular mechanisms underlying effects of ASV in IPF, we explored the truth using bioinformatics, plasmid construction, immunofluorescence staining, western blotting and other experiments. Dual luciferase reporter assay and bioinformatics proved that miR-200c not only acts as an upstream regulatory miRNA of ZEB1 but also has binding sites for the lncRNA-ATB. In A549 cell-based EMT models, ASV reduced the expression of lncRNA-ATB and upregulated miR-200c. Furthermore, overexpression of lncRNA-ATB and silencing of miR-200c reversed the down-regulation of ZEB1 and the inhibition of EMT processes by ASV. In addition, the intervention of ASV prevented lncRNA-ATB as a ceRNA from regulating the expression of ZEB1 through sponging miR-200c. Taken together, the results showed that ASV inhibited the EMT process through the lncRNA-ATB/miR-200c/ZEB1 signaling pathway, which provides a novel approach to the treatment of IPF.

Knockdown of ZEB1 Inhibits Hypertrophic Scarring through Suppressing the Wnt/β-catenin Signaling Pathway in a Mouse Model.

Hypertrophic scars (HS) cause functional impairment and cosmetic deformities following surgeries or burns (30% to 94%). There is no target therapy yet because the pathogenesis of HS progression is not well-known. In tissue fibrosis, Zinc finger E-box binding homeobox 1 (ZEB1) abnormal upregulation is an important cause for extracellular matrix (ECM) overexpression, which is the main molecular change in HS. Therefore, we hypothesized that ZEB1-knockdown inhibits HS formation. ZEB1 expression in human HS and TGF-β1-induced fibroblasts were identified by PCR and western blotting. ZEB1 was knockdown by siRNA in HS fibroblasts (HSFs) and mouse HS model (C57/BL6, male, 8-12 weeks). After 8-hour-transfection, HSFs were subjected to PCR, western blotting and CCK-8, apoptosis, migration and contraction assays. Mice HS were analyzed by HE staining, PCR and western blotting after 56 days. ZEB1 was upregulated in HS tissue (2.0-fold; p < 0.001). ZEB1 knockdown inhibited HSFs activity (0.6 to 0.7-fold; p < 0.001), the expression of fibrotic markers (0.4 to 0.6-fold; p < 0.001) and β-catenin, cyclinD1 and c-Myc expression (0.5-fold; p < 0.001). In mouse HS models, HS skin thickness was thinner (1.60 ± 0.40 mm vs. 4.04 ± 0.36 mm; p < 0.001) after ZEB1 knockdown. Knockdown of ZEB1 inhibits HS formation both in vitro and in vivo. However, this is an in vitro/mouse model and more validation is needed. The discovery of ZEB1 as a mediator of HS formation might be a potential therapeutic target in HS treatment.

MiR-186 regulates epithelial–mesenchymal transformation to promote nasopharyngeal carcinoma metastasis by targeting ZEB1

ObjectivesNasopharyngeal carcinoma (NPC) is an aggressive epithelial cancer. The expression of miR-186 is decreased in a variety of malignancies and can promote the invasion and metastasis of cancer cells. This study aimed to explore the role and possible mechanism of miR-186 in the metastasis and epithelial–mesenchymal transformation (EMT) of NPC. MethodsThe expression of miR-186 in NPC tissues and cells was detected by RT-PCR. Then, miR-186 mimic was used to transfect NPC cell lines C666-1 and CNE-2, and cell activity, invasion and migration were detected by CCK8, transwell and scratch assay, respectively. The expression of EMT-related proteins was analyzed by western blotting analysis. The binding relationship between miR-186 and target gene Zinc Finger E-Box Binding Homeobox 1 (ZEB1) was confirmed by double luciferase assay. ResultsThe expression of miR-186 in NPC was significantly decreased, and transfection of miR-186 mimic could significantly inhibit the cell activity, invasion, and migration, and regulate the protein expressions of E-cadherin, N-cadherin and vimentin in C666-1 and CNE-2 cells. Further experiments confirmed that miR-186 could directly target ZEB1 and negatively regulate its expression. In addition, ZEB1 has been confirmed to be highly expressed in NPC, and inhibition of ZEB1 could inhibit the activity, invasion, metastasis and EMT of NPC cells. And co-transfection of miR-186 mimic and si-ZEB1 could further inhibit the proliferation and metastasis of NPC. ConclusionmiR-186 may inhibit the proliferation, metastasis and EMT of NPC by targeting ZEB1, and the miR-186/ZEB1 axis plays an important role in NPC.

Glucocorticoid receptor regulates the epithelial-mesenchymal transition process through GR/ZEB1/E-cad and is involved in breast cancer endocrine drug resistance-a bioinformatics analysis.

Studies have shown that there is a connection between estrogen receptor (ER) and glucocorticoid receptor (GR), which can impact the epithelial-mesenchymal transition (EMT) process and contribute to endocrine resistance in breast cancer. However, the specific mechanism is unclear. It is crucial to investigate this mechanism further. This study aimed to confirm the role of GR in breast cancer endocrine resistance. Based on our hypothesis, GR is linked to a gene involved in the EMT process, and thus contributes to endocrine resistance in breast cancer. We obtained survival data and GR expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Additionally, we gathered GR expression data from Gene Expression Omnibus (GEO). Using Cytoscape, we constructed a protein-protein interaction (PPI) network and identified key genes. Data of Vimentin, E-cad, and Wnt/β-catenin expression were obtained from The Cancer Genome Atlas (TCGA). We used the co-expression method to identify key proteins. UALCAN and cBioPortal were utilized to verify the function of the key protein. In ER+ breast cancer, GR (P=3.12780899271121E-08) and zinc finger E-box binding homeobox 1 (ZEB1) (P=1.716157E-01) were lowly expressed and down-regulated genes of GR differentially expressed genes were enriched in cell adhesion molecules. We screened for the key protein ZEB1 and found high levels of it was positively associated with prolonged recurrence-free survival (RFS) in patients receiving endocrine therapy (P=0.0024), while high levels of E-cad were negatively associated (P=0.0038). GR expression was positively associated with ZEB1 (Spearman =0.29, P=8.50e-21), negatively associated with E-cad (Spearman =-0.13, P=5.17e-5), and negatively associated with the SETD1B (Spearman =-0.14, P=1.527e-5), a gene downstream of ZEB1. In contrast, ZEB1 expression was negatively correlated with E-cad (Spearman =-0.081, P=3.132e-3) and negatively correlated with SET domain-containing 1B (SETD1B) (Spearman =-0.177, P=9.07e-11). In ER+ breast cancers, GR expression is suppressed, and the EMT process is inhibited by suppressing ZEB1 expression and thus promoting E-cad expression. For the investigation of endocrine medication resistance in breast cancer, it is crucial to identify the mechanisms by how GR participates in the EMT process.

BCAT2 promotes melanoma progression by activating lipogenesis via the epigenetic regulation of FASN and ACLY expressions.

Melanoma is the most lethal skin cancer originating from the malignant transformation of epidermal melanocyte. The dysregulation of cellular metabolism is a hallmark of cancer, including in melanoma. Aberrant branched-chain amino acids (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Herein, we reported that the critical BCAA metabolism enzyme branched-chain amino acid transaminase 2 (BCAT2) is an oncogenic factor in melanoma by activating lipogenesis via the epigenetic regulation of fatty acid synthase (FASN) and ATP-citrate lyase (ACLY) expressions. Firstly, we found that BCAT2 expression was prominently increased in melanoma, and highly associated with clinical stage. Then, it was proved that the deficiency of BCAT2 led to impaired tumor cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Further, RNA sequencing technology and a panel of biochemical assays demonstrated that BCAT2 regulated de novo lipogenesis via the regulation of the expressions of both FASN and ACLY. Mechanistically, the inhibition of BCAT2 suppressed the generation of intracellular acetyl-CoA, mitigating P300-dependent histone acetylation at the promoter of FASN and ACLY, and thereby their transcription. Ultimately, zinc finger E-box binding homeobox 1 (ZEB1) was identified as the upstream transcriptional factor responsible for BCAT2 up-regulation in melanoma. Our results demonstrate that BCAT2 promotes melanoma progression by epigenetically regulating FASN and ACLY expressions via P300-dependent histone acetylation. Targeting BCAT2 could be exploited as a promising strategy to restrain tumor progression in melanoma.

ZEB1 potentiates chemoresistance in breast cancer stem cells by evading apoptosis.

Chemoresistance renders a challenge to the clinics to treat breast cancer patients. Current treatment strategies are effective in mitigating tumor growth but remain largely ineffective against cancer-initiating cells or breast Cancer Stem Cells (CSCs). Epithelial-to-mesenchymal-transition (EMT) regulates breast CSC physiology. Zinc finger E-box binding homeobox 1 (ZEB1) is a key EMT-transcription factor that regulates breast CSC - differentiation and metastasis. However, its potential role in modulating tumor chemoresistance has not yet been fully understood. In-silico analysis revealed a higher ZEB1 expression in breast cancer patients that leads to decreased overall and relapse-free survival. We generated sorted breast CSC with stable ZEB1 overexpression (CD24-/CD44+GFP-ZEB1) and/or silencing (CD24-/CD44+ZEB1 shRNA) as well as breast cancer cells with stable ZEB1 overexpression (CD24+GFP-ZEB1) and/or silencing (CD24+ZEB1 shRNA). An increased colony-forming efficiency and doxorubicin accumulation correlated with decreased promoter activity and expression profile of ABCC1 drug-efflux ABC transporter in CD24-/CD44+GFP-ZEB1. Additionally, CD24-/CD44+GFP-ZEB1 demonstrated doxorubicin-induced higher anti-apoptotic and lower pro-apoptotic protein expressions in the mitochondrial and cytosolic fractions. Chemoresistant CD24-/CD44+GFP-ZEB1 cells depicted 1000-fold higher IC-50 values of doxorubicin and decreased activation of JNK-p38 stress kinase molecular signaling-dependent mammosphere forming efficiency to evade apoptosis. Thus, ZEB1 and its downstream effectors are plausible therapeutic targets for the mitigation of breast cancer chemoresistance in patients.

Zeb1 Regulates the Function of Lympho-Myeloid Primed Progenitors after Transplantation.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal (EMT) transcription factor, acts as a critical regulator of hematopoietic stem cell (HSC) self-renewal and multi-lineage differentiation. Whether Zeb1 directly regulates the function of multi-potent progenitors primed for hematopoietic lineage commitment remains ill defined. By using an inducible Mx-1 Cre conditional mouse model where Zeb1 was genetically engineered to be deficient in the adult hematopoietic system (hereafter Zeb1-/-), we found that the absolute cell number of immunophenotypically defined lympho-myeloid primed progenitors (LMPPs) from Zeb1-/- mice was reduced. Myeloid- and lymphoid-biased HSCs in Zeb1-/- mice were unchanged, implying that defective LMPP generation from Zeb1-/- mice was not directly caused by an imbalance of lineage-biased HSCs. Functional analysis of LMPP from Zeb1-/- mice, as judged by competitive transplantation, revealed an overall reduction in engraftment to hematopoietic organs over 4 weeks, which correlated with minimal T-cell engraftment, reduced B-cell and monocyte/macrophage engraftment, and unperturbed granulocyte engraftment. Thus, Zeb1 regulates LMPP differentiation potential to select lympho-myeloid lineages in the context of transplantation.

A logical model of Ewing sarcoma cell epithelial-to-mesenchymal transition supports the existence of hybrid cellular phenotypes.

Ewing sarcoma (ES) is a highly aggressive pediatric tumor driven by the RNA-binding protein EWS (EWS)/friend leukemia integration 1 transcription factor (FLI1) chimeric transcription factor, which is involved in epithelial-mesenchymal transition (EMT). EMT stabilizes a hybrid cell state, boosting metastatic potential and drug resistance. Nevertheless, the mechanisms underlying the maintenance of this hybrid phenotype in ES remain elusive. Our study proposes a logical EMT model for ES, highlighting zinc finger E-box-binding homeobox 2 (ZEB2), miR-145, and miR-200 circuits that maintain hybrid states. The model aligns with experimental findings and reveals a previously unknown circuit supporting the mesenchymal phenotype. These insights emphasize the role of ZEB2 in the maintenance of the hybrid state in ES.

Expression of circular RNAs in myelodysplastic neoplasms and their association with mutations in the splicing factor gene SF3B1.

Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses,weproposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.

The study of long noncoding RNA TUG 1 and ZEB2-AS1 expression in newly diagnosed Egyptian adult acute myeloid leukemia patients

BackgroundThe hematopoietic malignancy acute myeloid leukemia is a fatal disease with poor clinical prognoses. Long non-coding RNA taurine-upregulated gene1 (lncRNA TUG1) and zinc finger E-box binding homeobox 2 antisense RNA1 (lncRNA ZEB2-AS1) are reported to participate in the development and progression of different types of malignancies. The goal of the current study was to evaluate the prognostic value of the lncRNAs TUG1 and ZEB2-AS1 as well as their various expression patterns in newly diagnosed Egyptian adult acute myeloid leukemia patients.MethodsWe assessed the expression levels of both lncRNA TUG1 and lncRNA ZEB2-AS1 using the quantitative real-time reverse transcription polymerase chain reaction technique (qRT-PCR) in 80 newly diagnosed AML patients and 20 healthy subjects.ResultslncRNA TUG1 expression was significantly higher in the AML cases compared to the controls (P < 0.001), whereas lncRNA ZEB2-AS1 expression was considerably lower in the AML cases in comparison with the controls (P < 0.001). The expression levels of the lncRNAs ZEB2-AS1 and TUG1 exhibited a significantly positive association in the AML group (P < 0.001). There was no difference in overall survival (OS) and disease-free survival (DFS) between the groups with low and high lncRNA TUG1 expression (P = 0.139 and 0.918, respectively). Furthermore, the AML cases with higher lncRNA ZEB2-AS1 expression levels had shorter DFS than patients with lower lncRNA ZEB2-AS1 expression levels (P = 0.014), while OS did not significantly differ between the studied cases with lower and higher lncRNA ZEB2-AS1 expression (P = 0.589).ConclusionOverexpression of lncRNA TUG1 could serve as a diagnostic biomarker for Egyptian adult AML cases, while lncRNA ZEB2-AS1 high expression could be regarded as an indicator of poor outcome in Egyptian adult AML studied cases.

ZEB1 interferes with human periodontal ligament stem cell proliferation and differentiation.

Periodontitis can eventually contribute to tooth loss. Zinc finger E-box binding homeobox 1 (ZEB1) is identified as overexpressed in the gingival tissue of mice with periodontitis. This study is designed to decipher the mechanism of ZEB1's involvement in periodontitis. Human periodontal mesenchymal stem cells (hPDLSCs) were exposed to LPS to mimic the inflammation in periodontitis. Following ZEB1 silencing, FX1 (an inhibitor of Bcl-6) treatment or ROCK1 overexpression, cell viability, and apoptosis were analyzed. Alkaline phosphatase (ALP) staining, Alizarin red staining, RT-qPCR, and western blot were performed to evaluate osteogenic differentiation and mineralization. hPDLSCs were processed for luciferase reporter assay and ChIP-PCR to confirm the association between ZEB1 and ROCK1. The induction of ZEB1 silencing resulted in reduced cell apoptosis, enhanced osteogenic differentiation, and mineralization. Nevertheless, these effects were significantly blunted by FX1. ZEB1 was confirmed to bind to the promoter sites of ROCK1 and regulate the ROCK1/AMPK. Whereas ROCK1 overexpression reversed the effects of ZEB1 silencing on Bcl-6/STAT1, as well as cell proliferation and osteogenesis differentiation. hPDLSCs displayed decreased proliferation and weakened osteogenesis differentiation in response to LPS. These impacts were mediated by ZEB1 regulating Bcl-6/STAT1 via AMPK/ROCK1.

Vitamin D deficiency contributes to the diabetic kidney disease progression via increase ZEB1/ZEB2 expressions

BackgroundDiabetic kidney disease (DKD) remains one of the main causes of end-stage renal disease (ESRD) and mortality in diabetic patients worldwide. Vitamin D deficiency (VitDD) is one of the main consequences of different chronic kidney disease (CKD) types and is associated with rapid progression to ESRD. Nevertheless, the mechanisms that lead to this process are poorly understood. This study aimed to characterize a model of diabetic nephropathy progression in VitDD and the epithelial-mesenchymal-transition (EMT) role in these processes.MethodsWistar Hannover rats received a diet with or without VitD before type 1 diabetes (T1D) induction. After this procedure, the rats were accompanied for 12 and 24 weeks after T1D induction and the renal function, structure, cell transdifferentiating markers and zinc finger e-box binding homeobox 1/2 (ZEB1/ZEB2) contribution to kidney damage were evaluated during the DKD progression.ResultsThe results showed an increase in glomerular tuft, mesangial and interstitial relative areas and renal function impairment in VitD-deficient diabetic rats compared to diabetic rats that received a VitD-containing diet. These alterations can be associated with increased expression of EMT markers, ZEB1 gene expression, ZEB2 protein expression and TGF-β1 urinary excretion. Decreased miR-200b expression, an important post-transcriptional regulator of ZEB1 and ZEB2 was also observed.ConclusionOur data demonstrated that VitD deficiency contributes to the rapid development and progression of DKD in diabetic rats induced by increase ZEB1/ZEB2 expressions and miR-200b downregulation.

Zeb1 and Tle3 are trans-factors that differentially regulate the expression of myosin heavy chain-embryonic and skeletal muscle differentiation.

Myosin heavy chain-embryonic encoded by the Myh3 gene is a skeletal muscle-specific contractile protein expressed during mammalian development and regeneration, essential for proper myogenic differentiation and function. It is likely that multiple trans-factors are involved in this precise temporal regulation of Myh3 expression. We identify a 4230 bp promoter-enhancer region that drives Myh3 transcription in vitro during C2C12 myogenic differentiation and in vivo during muscle regeneration, including sequences both upstream and downstream of the Myh3 TATA-box that are necessary for complete Myh3 promoter activity. Using C2C12 mouse myogenic cells, we find that Zinc-finger E-box binding homeobox 1 (Zeb1) and Transducin-like Enhancer of Split 3 (Tle3) proteins are crucial trans-factors that interact and differentially regulate Myh3 expression. Loss of Zeb1 function results in earlier expression of myogenic differentiation genes and accelerated differentiation, whereas Tle3 depletion leads to reduced expression of myogenic differentiation genes and impaired differentiation. Tle3 knockdown resulted in downregulation of Zeb1, which could be mediated by increased expression of miR-200c, a microRNA that binds to Zeb1 transcript and degrades it. Tle3 functions upstream of Zeb1 in regulating myogenic differentiation since double knockdown of Zeb1 and Tle3 resulted in effects seen upon Tle3 depletion. We identify a novel E-box in the Myh3 distal promoter-enhancer region, where Zeb1 binds to repress Myh3 expression. In addition to regulation of myogenic differentiation at the transcriptional level, we uncover post-transcriptional regulation by Tle3 to regulate MyoG expression, mediated by the mRNA stabilizing Human antigen R (HuR) protein. Thus, Tle3 and Zeb1 are essential trans-factors that differentially regulate Myh3 expression and C2C12 cell myogenic differentiation in vitro.

Trophoblast cell surface antigen-2 phosphorylation triggered by binding of galectin-3 drives metastasis through down-regulation of E-cadherin

The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of β-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of β-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7cells and DU145cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility invivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.

Identification of a novel therapeutic target for lung cancer: Mitochondrial ribosome protein L9

Lung cancer has a high fatality rate and incidence rate. At present, the initial and progress mechanism of lung cancer has not been completely elucidated and new therapeutic targets still need to be developed. In this study, the screening process was based on lung cancer expression profile data and survival analysis. Mitochondrial ribosome protein L9 (MRPL9) was upregulated in lung cancer tissues and related to the poor overall survival rate and recurrence-free survival rate of lung cancer patients. Knockdown of MRPL9 inhibited the proliferation, sphere-formation, and migration ability of lung cancer cells. MRPL9 was associated with the c-MYC signaling pathway, and lung cancer patients with high expression of both MRPL9 and MYC had a poor prognosis. Furthermore, c-MYC was associated with the epithelial-mesenchymal transition (EMT) regulatory protein zinc finger E-box binding homeobox 1 (ZEB1) by bioinformatics analysis. The relationship between ZEB1 and c-MYC was further confirmed by interfering with c-MYC expression. MRPL9 is a potential therapeutic target for lung cancer and exerts its biological functions by affecting the transcription factor c-MYC thereby regulating the EMT regulator ZEB1.

Long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense RNA 1 regulates myocardial fibrosis in diabetes through the Hippo-Yes-associated protein signaling pathway.

Fibrosis is the principle reason for heart failure in diabetes. Regarding the involvement of long non-coding ribonucleic acid zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) in diabetic myocardial fibrosis, we explored its specific mechanism. Human cardiac fibroblasts (HCF) were treated with high glucose (HG) and manipulated with plasmid cloning deoxyribonucleic acid 3.1-ZEB1-AS1/microribonucleic acid (miR)-181c-5p mimic/short hairpin RNA specific to sirtuin 1 (sh-SIRT1). ZEB1-AS1, miR-181c-5p expression patterns, cell viability, collagen I and III, α-smooth muscle actin (α-SMA), fibronectin levels and cell migration were assessed by reverse transcription quantitative polymerase chain reaction, cell counting kit-8, western blot and scratch tests. Nuclear/cytosol fractionation assay verified ZEB1-AS1 subcellular localization. The binding sites between ZEB1-AS1 and miR-181c-5p, and between miR-181c-5p and SIRT1 were predicted and verified by Starbase and dual-luciferase assays. The binding of SIRT1 to Yes-associated protein (YAP) and YAP acetylation levels were detected by co-immunoprecipitation. Diabetic mouse models were established. SIRT1, collagen I, collagen III, α-SMA and fibronectin levels, mouse myocardium morphology and collagen deposition were determined by western blot, and hematoxylin-eosin and Masson trichrome staining. Zinc finger E-box binding homeobox 1 antisense 1 was repressed in HG-induced HCFs. ZEB1-AS1 overexpression inhibited HG-induced HCF excessive proliferation, migration and fibrosis, and diminished collagen I, collagen III, α-SMA and fibronectin protein levels in cells. miR-181c-5p had targeted binding sites with ZEB1-AS1 and SIRT1. SIRT1 silencing/miR-181c-5p overexpression abrogated ZEB1-AS1-inhibited HG-induced HCF proliferation, migration and fibrosis. ZEB1-AS1 suppressed HG-induced HCF fibrosis through SIRT1-mediated YAP deacetylation. ZEB1-AS1 and SIRT1 were repressed in diabetic mice, and miR-181c-5p was promoted. ZEB1-AS1 overexpression improved myocardial fibrosis in diabetic mice, and reduced collagen I, collagen III, α-SMA and fibronectin protein levels in myocardial tissues. Long non-coding ribonucleic acid ZEB1-AS1 alleviated myocardial fibrosis through the miR-181c-5p-SIRT1-YAP axis in diabetic mice.

The Conserved LncRNA DIO3OS Restricts Hepatocellular Carcinoma Stemness by Interfering with NONO-Mediated Nuclear Export of ZEB1 mRNA.

Hepatocellular carcinoma (HCC) is an aggressive and fatal disease caused by a subset of cancer stem cells (CSCs). It is estimated that there are approximately 100000 long noncoding RNAs (lncRNAs) in humans. However, the mechanisms by which lncRNAs affect tumor stemness remain poorly understood. In the present study, it is found that DIO3OS is a conserved lncRNA that is generally downregulated in multiple cancers, including HCC, and its low expression correlates with poor clinical outcomes in HCC. In in vitro cancer cell lines and an in vivo spontaneous HCC mouse model, DIO3OS markedly represses tumor development via its suppressive role in CSCs through downregulation of zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, DIO3OS represses ZEB1 post-transcriptionally without affecting its mRNA levels. Subsequent experiments show that DIO3OS interacts with the NONO protein and restricts NONO-mediated nuclear export of ZEB1 mRNA. Overall, these findings demonstrate that the DIO3OS-NONO-ZEB1 axis restricts HCC development and offers a valuable candidate for CSC-targeted therapeutics for HCC.

LincRNA PRNCR1 activates the Wnt/β-catenin pathway to drive the deterioration of hepatocellular carcinoma via regulating miR-411-3p/ZEB1 axis

ABSTRACT Hepatocellular carcinoma (HCC) is an intractable malignant disease with high incidence rate annually. LincRNA PRNCR1 has been confirmed as a tumor supporter, while its functions in HCC remain unclear. This study aims to explore the mechanism of LincRNA PRNCR1 in hepatocellular carcinoma. The qRT-PCR was applied to the quantification of non-coding RNAs. Cell counting Kit-8 (CCK-8), Transwell assay and flow cytometry assay were applied to reflect the change in the phenotype of HCC cells. Moreover, the databases including Targetscan and Starbase and dual-luciferase reporter assay were applied to investigate the interaction of the genes. The western blot was applied to detect the abundance of proteins and the activity of the related pathways. Elevated LincRNA PRNCR1 was dramatically upregulated in HCC pathological samples and cell lines. MiR-411-3p served as a target of LincRNA PRNCR1, and decreased miR-411-3p was found in the clinical samples and cell lines. LincRNA PRNCR1 downregulation could induce the expression of miR-411-3p, and LincRNA PRNCR1 silence could impede the malignant behaviors via increasing the abundance of miR-411-3p. Zinc finger E-box binding homeobox 1 (ZEB1) was confirmed as a target of miR-411-3p, which remarkably upregulated in HCC cells, and ZEB1 upregulation could significantly rescue the effect of miR-411-3p on malignant behaviors of HCC cells. Moreover, LincRNA PRNCR1 was confirmed to involve the Wnt/β-catenin pathway via regulating miR-411-3p/ZEB1 axis. This study suggested that LincRNA PRNCR1 could drive the malignant progression of HCC via regulating miR-411-3p/ZEB1 axis.