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Polo-like kinase 1 (PLK1) is a serine/threonine–protein kinase essential for regulating multiple stages of cell cycle progression in mammals. Aberrant regulation of PLK1 has been observed in numerous human cancers and is linked to poor prognoses. However, its role in the pathogenesis of colorectal cancer (CRC) in the Middle East remains unexplored. PLK1 overexpression was noted in 60.3% (693/1149) of CRC cases and was significantly associated with aggressive clinico-pathological parameters and p-ERK1/2 overexpression. Intriguingly, multivariate logistic regression analysis identified PLK1 as an independent predictor of lymph node metastasis. Our in vitro experiments demonstrated that CRC cells with high PLK1 levels were resistant to 5-Fu treatment, while those with low PLK1 expression were sensitive. To investigate PLK1′s role in chemoresistance, we used the specific inhibitor volasertib, which effectively reversed 5-Fu resistance. Interestingly, forced PLK1 expression activated the CRAF-MEK-ERK signaling cascade, while its inhibition suppressed this cascade. PLK1 knockdown reduced epithelial-to-mesenchymal transition (EMT) progression and stem cell-like traits in 5-Fu-resistant cells, implicating PLK1 in EMT induction and stemness in CRC. Moreover, silencing ERK1/2 significantly mitigated chemoresistance, EMT, and stemness properties in CRC cell lines that express PLK1. Furthermore, the knockdown of Zeb1 attenuated EMT and stemness, suggesting a possible link between EMT activation and the maintenance of stemness in CRC. Our findings underscore the pivotal role of PLK1 in mediating chemoresistance and suggest that PLK1 inhibition may represent a potential therapeutic strategy for the management of aggressive colorectal cancer subtypes. [ABSTRACT FROM AUTHOR]

Luteinizing hormone (LH), a heterodimeric glycoprotein produced by pituitary gonadotrope cells, regulates gonadal function. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates LH synthesis and secretion. GnRH induces LHβ subunit (Lhb) expression via the transcription factor, early growth response 1 (EGR1), acting on the Lhb promoter. In contrast, overexpression of zinc finger E-box binding homeobox 1 (ZEB1) represses LH production in mice, but the underlying mechanism was not previously elucidated. Here, we observed that ZEB1 inhibited GnRH-stimulated but not basal Lhb mRNA expression in homologous murine LβT2 cells. Moreover, ZEB1 blocked GnRH and/or EGR1 induction of murine Lhb but not human LHB promoter-reporter activity in these cells. Using chimeric reporters, we mapped the species-specific ZEB1 sensitivity to sequence differences, including in Z- and E-boxes, in the proximal Lhb/LHB promoters, immediately upstream of the transcription start sites. ZEB1 bound to the murine Lhb promoter with higher affinity than to the human LHB promoter in this region. To examine ZEB1's physiological role in LH synthesis, we characterized gonadotrope-specific Zeb1 knockout mice. Loss of ZEB1 in gonadotropes did not affect LH production or secretion. Collectively, the data suggest that ZEB1, when overexpressed, can inhibit GnRH/EGR1 induction of murine Lhb transcription but does not play a necessary role in LH synthesis in mice. [ABSTRACT FROM AUTHOR]

Simple Summary: Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer progression, contributing to the invasive and migratory abilities of tumor cells. In this study, we show that FBXO11 promotes the degradation of ZEB1, a key EMT regulator, via ubiquitination. Loss of FBXO11 increases ZEB1 levels, enhancing the invasiveness of lung cancer cells, while its overexpression reduces ZEB1 and suppresses invasion. Importantly, higher FBXO11 expression is associated with better prognosis in non-small cell lung cancer (NSCLC), highlighting its potential role as a therapeutic target for controlling EMT and cancer metastasis. Epithelial-to-mesenchymal transition (EMT) affects the invasion and migration of cancer cells. Here, we show that FBXO11 recognizes and promotes ubiquitin-mediated degradation of ZEB1. There is a strong association between FBXO11 and ZEB1 in non-small cell lung cancer (NSLC) in a clinical database. FBXO11 interacts with ZEB1, a core inducer of EMT. FBXO11 leads to increased ubiquitination and proteasomal degradation of ZEB1. Depletion of endogenous FBXO11 causes ZEB1 protein accumulation and EMT in A549 and H1299 cells, while overexpression of FBXO11 reduces ZEB1 protein abundance and cellular invasiveness. Importantly, the depletion of ZEB1 suppresses the increased migration and invasion of A549 and H1299 cells promoted by the depletion of FBXO11. The same results are shown in xenograft tumors. High FBXO11 expression is associated with a favorable prognosis in NSLC. Collectively, our study demonstrates that FBXO11 modulates EMT by mediating the stability of ZEB1 in lung cancer cells. [ABSTRACT FROM AUTHOR]

During the development of the mouse dentate gyrus (DG), granule neuronal progenitors (GNPs) arise from glial fibrillary acidic protein (GFAP)-expressing neural stem cells in the dentate notch. However, the transcriptional regulators that control their stepwise differentiation remain poorly defined. Since neurogenesis involves epithelial-to-mesenchymal transition (EMT)-like processes, we investigated the spatio-temporal expression profiles of the EMT transcription factors Zeb1, Scratch2 (Scrt2) and Nkx6-2 in relation to known GNP markers. Our results show that Zeb1 and Scrt2 exhibit sequential, but partially overlapping expression across embryonic and postnatal stages of GNP differentiation. Zeb1 is highly enriched in gfap-GFP+/Sox2+ neural stem/ progenitor pools and subsets of Tbr2+/Prox1+/NeuroD+ intermediate GNPs, whereas Scrt2 predominates in Tbr2+/Prox1+/NeuroD+ GNPs. Strikingly, the neuronal EMT regulator Nkx6-2 shows selective expression in postnatal Tbr2+/ Prox1+ GNPs, but it is excluded from embryonic counterparts. This temporally coordinated yet distinct expression of Zeb1, Scrt2 and Nkx6-2 reveals discrete transcriptional programs orchestrating GNP differentiation and neurogenic progression at embryonic versus postnatal stages of DG neurogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Although microRNA (miR)-150-5p participates in the progression of renal fibrosis, its mechanism of action remains elusive. Methods: A mouse model of unilateral ureteral obstruction was used. The in vitro renal fibrosis model was established by stimulating human kidney 2 (HK-2) cells with transforming growth factor beta 1 (TGF-β1). The expression profiles of miR-150-5p, zinc finger E-box binding homeobox 1 (ZEB1), and other fibrosis- and epithelial-mesenchymal transition (EMT)-linked proteins were determined using Western blot and quantitative reverse transcription polymerase chain reaction. The relationship between miR-150-5p and ZEB1 in HK-2 cells was confirmed by a dual-luciferase reporter assay. Results: Both in vivo and in vitro renal fibrosis models revealed reduced miR-150-5p expression and elevated ZEB1 level. A significant decrease in E-cadherin levels, as well as increases in alpha smooth muscle actin (α-SMA) and collagen type I (Col-I) levels, was seen in TGF-β1-treated HK-2 cells. The overexpression of miR-150-5p ameliorated TGF-β1-mediated fibrosis and EMT. Notably, miR-150-5p acts by directly targeting ZEB1. A significant reversal of the inhibitory impact of miR-150-5p on TGF-β1-mediated fibrosis and EMT in HK-2 cells was observed upon ZEB1 overexpression. Conclusion: MiR-150-5p suppresses TGF-β1-induced fibrosis and EMT by targeting ZEB1 in HK-2 cells, providing helpful insights into the therapeutic intervention of renal fibrosis. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is an aggressive primary intestinal malignancy with the third-highest incidence and second-highest mortality among all cancer types worldwide. Transcription factors (TFs) regulate cell development and differentiation owing to their ability to recognize specific DNA sequences upstream of genes. Numerous studies have demonstrated a strong correlation between TFs, the etiology of tumors, and therapeutic approaches. Here, we aimed to explore prognosis-related TFs and comprehend their carcinogenic mechanisms, thereby offering novel insights into the diagnosis and management of CRC. Materials and Methods: Differentially expressed TFs between CRC and normal tissues were identified leveraging The Cancer Genome Atlas database, Weighted correlation network analysis and Cox regression analysis were performed to identify prognosis-related TFs. The cellular functions of hub TF zinc finger E-box binding homeobox 1 (ZEB1) were determined using by 5-ethynyl-2'-deoxyuridine and cell invasion assays in CRC cells. RNAsequencing, Kyoto Encyclopedia of Genes and Genomes enrichment, and gene set enrichment analyses were used to identify the cellular processes in which ZEB1 participates. Immunoaffinity purification, silver staining mass spectrometry, and a chromatin immunoprecipitation assay were conducted to search for proteins that might interact with ZEB1 and the target genes they jointly regulate. Results: Thirteen central TFs related to prognosis were identified through bioinformatics analysis techniques. Among these TFs, ZEB1 emerged as the TF most closely associated with CRC, as determined through a combination of regulatory network diagrams, survival curves, and phenotype analyses. ZEB1 promotes CRC cell growth by recruiting the NuRD(MTA1) complex, and the ZEB1/NuRD(MTA1) complex transcriptionally represses glycolysis-associated tumor suppressor genes. Conclusion: Our study not only identified a hub biomarker related to CRC prognosis but also revealed the specific molecular mechanisms through which ZEB1 affects cancer progression. These insights provide crucial evidence for the diagnosis of CRC and potential treatment opportunities. [ABSTRACT FROM AUTHOR]

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Background The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. Methods We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Results Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus -infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli , induced EMT-like effects in cell culture. Conclusions Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis. [ABSTRACT FROM AUTHOR]

Background: PD-L1 overexpression is commonly observed in various malignancies and is strongly correlated with poor prognoses for cancer patients. Moreover, PD-L1 has been shown to play a significant role in promoting angiogenesis and epithelial-mesenchymal transition (EMT) processes across different cancer types. Methods: The relationship between PD-L1 and vasculogenic mimicry as well as epithelial-mesenchymal transition (EMT) was explored by bioinformatics approach and immunohistochemistry. The functions of PD-L1 in regulating the expression of ZEB1 and the EMT process were assessed by Western blotting and q-PCR assays. The impact of PD-L1 on the migratory and proliferative capabilities of A549 and H1299 cells was evaluated through wound healing, cell invasion, and CCK8 assays following siRNA-mediated PD-L1 knockdown. Tube formation assay was utilized to evaluate the presence of VM structures. Results: In this study, increased PD-L1 expression was observed in A549 and H1299 cells compared to normal lung epithelial cells. Immunohistochemical analysis revealed a higher prevalence of VM structures in the PD-L1-positive group compared to the PD-L1-negative group. Additionally, high PD-L1 expression was also found to be significantly associated with advanced TNM stage and increased metastasis. Following PD-L1 knockdown, NSCLC cells exhibited a notable reduction in their ability to form tube-like structures. Moreover, the levels of key EMT and VM-related markers, including N-cadherin, MMP9, VE-cadherin, and VEGFA, were significantly decreased, while E-cadherin expression was upregulated. In addition, the migration and proliferation capacities of both cell lines were significantly inhibited after PD-L1 or ZEB1 knockdown. Conclusions: Knockdown PD-L1 can inhibit ZEB1-mediated EMT, thereby hindering the formation of VM in NSCLC. [ABSTRACT FROM AUTHOR]

Objectives: Circular RNAs (circRNAs) serve a crucial regulatory role in ovarian cancer (OC). Circular RNA ArfGAP with FG repeats 1 (circAGFG1) has been shown to be involved in promoting the progression of several cancers, containing triple-negative breast cancer, esophageal cancer and colorectal cancer. However, the function of circAGFG1 in OC is unclear. Methods: Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted to determine the expression levels of circAGFG1 and miR-409-3p. The proliferation and metastasis of cells were determined by colony formation assays, EdU assays, transwell assays and wound healing assays. In addition, a dual-luciferase reporter assay was performed to validate the mechanism between circAGFG1, miR-409-3p, and ZEB1. Results: Our data suggested that circAGFG1 was significantly overexpressed in OC tissues compared to normal ovarian epithelial tissues. Overexpression of circAGFG1 was correlated with intraperitoneal metastasis, tumor recurrence and advanced stage. Additionally, circAGFG1 overexpression revealed a poor prognosis in OC patients. Knockdown of circAGFG1 suppressed the proliferation, invasion and migration of OC cells. Mechanistically, circAGFG1 acted as a sponge of miR-409-3p to enhance the expression level of zinc finger E-box binding homeobox 1 (ZEB1), thereby conferring OC cell proliferation, invasion and migration. Importantly, re-expression of ZEB1 effectively reversed the effects of circAGFG1 knockdown on OC cells. Conclusions: In summary, our study indicated that circAGFG1 may act as a prognostic biomarker and potential therapeutic target for patients with OC. [ABSTRACT FROM AUTHOR]

Background: MicroRNA-200b-3p (miR-200b-3p) plays a pivotal role in inflammatory responses and is implicated in various inflammatory disorders. In this study, we aim to explore the role of miR-200b-3p in the inflammatory response in heart failure (HF). Methods: Patients diagnosed with heart failure and age-matched healthy controls were studied. Peripheral blood samples from participants were collected for RNA-seq analysis to explore the expression profile of miR-200b-3p. The predictive value of miR-200b-3p and ZEB1 in the prognosis of heart failure was evaluated by analyzing the receiver operating characteristic (ROC) curve. Bioinformatics analysis and double luciferase reporter gene analysis were used to confirm the interaction between miR-200b-3p and ZEB1. Real-time quantitative polymerase chain reaction (QRT-PCR) was used to detect the expression levels of miR-200b-3p and ZEB1 in cardiopulmonary bypass. Additionally, the effects of miR-200b-3p on myocardial cell line (H9c2) injury were evaluated by enzyme-linked immunosorbent assay (ELISA). Results: In the extracardiac circulation of HF patients, miR-200b-3p expression was significantly reduced, while ZEB1 levels were notably elevated. Analysis of the ROC curve revealed that miR-200b-3p and ZEB1 have predictive value in the prognosis of HF patients. The double luciferase reporter experiment demonstrated that miR-200b-3p binds to ZEB1 and inhibits its expression. Overexpression of miR-200b-3p demonstrated a remarkable ability to alleviate inflammation and inhibit the damage to myocardial cells in vivo. Conclusion: MiR-200b-3p can target and inhibit ZEB1, reducing the inflammatory reaction of myocardial cells. The miR-200b-3p/ZEB1 network may be helpful in preventing and treating HF. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Background: Airway remodelling plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Epithelial–mesenchymal transition (EMT) is a significant process during the occurrence of airway remodelling. Increasing evidence suggests that glucose transporter 3 (GLUT3) is involved in the epithelial mesenchymal transition (EMT) process of various diseases. However, the role of GLUT3 in EMT in the airway epithelial cells of COPD patients remains unclear. Methods: We detected the levels of GLUT3 in the peripheral lung tissue of COPD patients and cigarette smoke (CS)-exposed mice. Two Gene Expression Omnibus GEO datasets were utilised to analyse GLUT3 gene expression profiles in COPD. Western blot and immunofluorescence were used to detect GLUT3 expression. In addition, we used the AAV9-GLUT3 inhibitor to reduce GLUT3 expression in the mice model. Masson's staining and lung function measurement were used detect the collagen deposition and penh in the mice. A cell study was performed to confirm the regulatory effect of GLUT3. Inhibition of GLUT3 expression with siRNA, Western blot, and immunofluorescence were used to detect the expression of E-cadherin, N-cadherin, vimentin, p65, and ZEB1. Results: Based on the GEO data set analysis, GLUT3 expression in COPD patients was higher than in non-smokers. Moreover, GLUT3 was highly expressed in COPD patients, CS exposed mice, and BEAS-2B cells treated with CS extract (CSE). Further research revealed that down-regulation of GLUT3 significantly alleviated airway remodelling in vivo and in vitro. Lung function measurement showed that GLUT3 reduction reduced airway resistance in experimental COPD mice. Mechanistically, our study showed that reduction of GLUT3 inhibited CSE-induced EMT by down-regulating the NF-κB/ZEB1 pathway. Conclusion: We demonstrate that CS enhances the expression of GLUT3 in COPD and further confirm that GLUT3 may regulate airway remodelling in COPD through the NF-κB/ZEB1 pathway; these findings have potential value in the diagnosis and treatment of COPD. The down-regulation of GLUT3 significantly alleviated airway remodelling and reduced airway resistance in vivo. Our observations uncover a key role of GLUT3 in modulating airway remodelling and shed light on the development of GLUT3-targeted therapeutics for COPD. [ABSTRACT FROM AUTHOR]

Background: Bladder cancer (BC) is one of the most common malignancies of the genitourinary system. Phosphofructokinase 1 (PFK-1) is one of member of PFK, which plays an important role in reprogramming cancer metabolism, such as lactylation modification. Zinc finger E-box-binding homeobox 1 (ZEB1) has been demonstrated to be a oncogene in many cancers. Therefore, this study was performed to explore the effects of PFK-1 on the lactylation of ZEB1 in BC development. Methods: Cell viability was measured using the CCK-8 kit. The glucose assay kit and lactate assay kit were used to detect glucose utilization and lactate production. The DNA was purified and quantified by qRT-PCR. Results: In the present study, we found that ZEB1 expression levels were significantly elevated in bladder cancer cells. Impaired PFK-1 expression inhibits proliferation, migration, and invasion of BC cells and suppresses tumour growth in vivo. We subsequently found that knockdown of PFK-1 decreases glycolysis, including reduced glucose consumption, lactate production and total extracellular acidification rate (ECAR). Mechanistically, PFK-1 inhibits histone lactylation of bladder cancer cells, and thus inhibits the transcription activity of ZEB1. Conclusion: Our results suggest that PFK-1 can inhibit the malignant phenotype of bladder cancer cells by mediating the lactylation of ZEB1. These findings suggested PFK-1 to be a new potential target for bladder cancer therapy. [ABSTRACT FROM AUTHOR]

Background: ZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression. Methods: We employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas. Results: Silencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression. Conclusions: Collectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future. [ABSTRACT FROM AUTHOR]

Cell migration is a crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial–mesenchymal transition (EMT), along with the changes in the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2), and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT proteins. Consequently, we have identified MHY9 (Myosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed to cell migration and the alteration in the expression of EMT marker proteins. Co-immunoprecipitation revealed the formation of a complex involving p-Tyr42 RhoA, PLD1, and MYH9. These proteins were found to be distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA PLD1 and MYH9 associate with the ZEB1 promoter. The suppression of ZEB1 mRNA levels was achieved through the knockdown of RhoA, PLD1, and MYH9 using si-RNAs. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration. [ABSTRACT FROM AUTHOR]

DPY30 belongs to the core subunit of components of the histone lysine methyltransferase complex, which is implicated in tumorigenesis, cell senescence, and other biological events. However, its contribution to colorectal carcinoma (CRC) progression and metastasis has yet to be elucidated. Therefore, this study aimed to investigate the biological function of DPY30 in CRC metastasis both in vitro and in vivo. Herein, our results revealed that DPY30 overexpression is significantly positively correlated with positive lymph nodes, epithelial-mesenchymal transition (EMT), and CRC metastasis. Moreover, DPY30 knockdown in HT29 and SW480 cells markedly decreased EMT progression, as well as the migratory and invasive abilities of CRC cells in vitro and lung tumor metastasis in vivo. Mechanistically, DPY30 increased histone H3K4me3 level and promoted EMT and CRC metastasis by upregulating the transcriptional expression of ZEB1. Taken together, our findings indicate that DPY30 may serve as a therapeutic target and prognostic marker for CRC. [ABSTRACT FROM AUTHOR]

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness. [ABSTRACT FROM AUTHOR]

Background: Epithelial ovarian cancer (EOC) is a pathological type with a higher mortality rate among gynecological cancers today. Long-chain noncoding RNAs (lncRNAs) can regulate the transcription and expression of cellular genes. However, the downstream molecules regulated by lncRNA HOTAIR have not been well studied. The effects of downregulated lncRNA HOTAIR on EOC invasiveness and tumorigenicity in nude mice, along with TGF- β1 and ZEB1 in epithelial ovarian cancer cells, need to be investigated in further research. Results: RT-qPCR was used to detect lncRNA HOTAIR and TGF-β1 and ZEB1 mRNA expression in EOC SKOV3 cells. The expression of lncRNA HOTAIR in SKOV3 cells transfected with the recombinant shHOTAIR interference plasmid was significantly lower than that of the negative control. Compared with the negative control, the matrix gel invasion ability of shHOTAIR SKOV3 cells in vitro and their tumorigenicity in nude mice were significantly reduced. Moreover, compared with the control, the expression of ZEB1 protein in shHOTAIR-SKOV3 xenograft tumors was significantly reduced. Downregulation of lncRNA HOTAIR expression significantly reduced TGF-β1 and ZEB1 mRNA expression, but increased the expression of E-cadherin mRNA. In summary, downregulated lncRNA HOTAIR in EOC SKOV3 cells transfected with shHOTAIR can inhibit TGF-β1, reduce ZEB1, increase E-cadherin, and significantly reduce the invasiveness and tumorigenicity of ovarian epithelial cancer SKOV3 cells. Conclusions: These results suggest that the lncRNA HOTAIR may be an effective target for the treatment of human EOC. [ABSTRACT FROM AUTHOR]

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype in women worldwide. The various alterations in the expression of different microRNAs (miRNAs) have been reported as crucial in the development of metastasis in breast tumors. Objectives: This study investigated the effect of sodium butyrate (NaB) on cell survival, cell metastasis and expression of miR-101, ZEB1, ZEB2 and E- cadherin in MDA-MB-468 cells as a TNBC cell line. Methods: Cell viability was evaluated using the (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay), and the metastasis potential of MDA-MB-468 cells was investigated using the scratch and transwell assay. The expression of genes involved in the metastasis process was measured using real-time polymerase chain reaction (PCR). Results: The MTT assay showed that NaB attenuated MDA-MB-468 cell survival dose-dependently with an IC50 value of 3.1 mM after 72 h treatment. The scratch and transwell assays also showed the anti-metastatic potential of NaB. The expression of miR-101, E-cadherin, ZEB1, and ZEB2 was significantly difference in MDA-MB-468 cells treated with 3.1 mM NaB after 72 hours (P < 0.05). E-cadherin and miR-101 were up-regulated, while the expression of ZEB1 and ZEB2 was significantly down-regulated compared to the untreated cells. This suggests that NaB increased cell attachment and prevented metastasis. In addition, NaB (IC50 value) restored the expression of miR-101, as a tumor suppressor, in MDA-MB-468 cells confirming its anti-cancer potency. Conclusions: Sodium butyrate can be used as a drug to suppress invasion and cell migration in TNBC cells. However, further studies are needed to demonstrate the putative anti-metastatic mechanism of NaB in preclinical and clinical settings. [ABSTRACT FROM AUTHOR]

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial–mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis. [ABSTRACT FROM AUTHOR]

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Cervical cancer (CC) is the most common type of gynecological malignancy in women, and targeting stem cells and inhibiting tumor stem cell-like properties of CC remains an important field of research as an attempt to improve treatment outcomes. This study focused on Ubiquitin-specific-processing Protease 43 (USP43), a member of the deubiquitinase (DUBs) family known to play a role in tumor progression, and analysis of the The Cancer Genome Atlas (TCGA) data and survival computations revealed that USP43 was highly expressed in CC and correlated with poor prognosis. However, the role of USP43 in CC has been under-reported, and its underlying mechanism remains unclear. To investigate the effect and mechanism of USP43, its expression in CC cells and tissues were examined, and the results showed that it was significantly upregulated. Subsequently, knockdown experiments revealed that reducing USP43 expression suppressed CC cell proliferation, and depleting USP43 inhibited the stem cell-like properties of CC cells and impaired their migration abilities. Further investigations indicated that USP43 promoted Zinc finger E-box binding protein 1 (ZEB1)-induced activation of Extracellular regulatory kinase 1/2 (ERK1/2) signaling in CC. Based on these findings, we propose that USP43 could serve as a promising target for CC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 100 000 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. [ABSTRACT FROM AUTHOR]

Neural precursors generate neurons in the embryonic brain and in restricted niches of the adult brain in a process called neurogenesis. The precise control of cell proliferation and differentiation in time and space required for neurogenesis depends on sophisticated orchestration of gene transcription in neural precursor cells. Much progress has been made in understanding the transcriptional regulation of neurogenesis, which relies on dose- and context-dependent expression of specific transcription factors that regulate the maintenance and proliferation of neural progenitors, followed by their differentiation into lineage-specified cells. Here, we review some of the most widely studied neurogenic transcription factors in the embryonic cortex and neurogenic niches in the adult brain. We compare functions of these transcription factors in embryonic and adult neurogenesis, highlighting biochemical, developmental, and cell biological properties. Our goal is to present an overview of transcriptional regulation underlying neurogenesis in the developing cerebral cortex and in the adult brain. [ABSTRACT FROM AUTHOR]

Epithelial‐mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor‐outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9‐mediated epigenetic editing, resulting in highly‐specific and nearly complete suppression of ZEB1 in vivo, accompanied by long‐lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9‐KRAB) enabled the discovery of a ZEB1‐dependent‐signature of 26 genes differentially‐expressed and ‐methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally‐spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1‐silencing are enriched in a subset of human breast tumors, illuminating a clinically‐relevant hybrid‐like state. Thus, the synthetic epi‐silencing of ZEB1 induces stable "lock‐in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome‐engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) circularized by back-splicing of pre-mRNA are widely expressed and affected the proliferation, invasion and metastasis of bladder cancer (BCa). However, the mechanism underlying circRNA biogenesis in mediating the distant metastasis of BCa still unexplored. Methods: RNA sequencing data between BCa and normal adjacent tissues was applied to identify the differentially expressed circRNAs. The functions of circNIPBL in BCa were investigated via a series of biochemical experiments. The Clinical significance of circNIPBL was examined in a cohort of larger BCa tissues. Results: In the present study, we identified a novel circRNA (hsa_circ_0001472), circNIPBL, which was significantly upregulated and had great influence on the poor prognosis of patients with BCa. Functionally, circNIPBL promotes BCa metastasis in vitro and in vivo. Mechanistically, circNIPBL upregulate the expression of Wnt5a and activated the Wnt/β-catenin signaling pathway via directly sponged miR-16-2-3p, leading to the upregulation of ZEB1, which triggers the EMT of BCa. Moreover, we revealed that ZEB1 interacted with the flanking introns of exons 2–9 on NIPBL pre-mRNA to trigger circNIPBL biogenesis, thus forming a positive feedback loop. Importantly, circNIPBL overexpression significantly facilitated the distant metastasis of BCa in the orthotopic bladder cancer model, while silencing ZEB1 remarkably blocked the effects of metastasis induced by circNIPBL overexpression. Conclusions: Our study highlights that circNIPBL-induced Wnt signaling pathway activation triggers ZEB1-mediated circNIPBL biogenesis, which forms a positive feedback loop via the circNIPBL/miR-16-2-3p/Wnt5a/ZEB1 axis, supporting circNIPBL as a novel therapeutic target and potential biomarker for BCa patients. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms underlying osteoclast differentiation provides insights into bone loss and even osteoporosis. The specific mechanistic actions of cullin 4A (CUL4A) in osteoclast differentiation and resultant osteoporosis is poorly explored. We developed a mouse model of osteoporosis using bilateral ovariectomy (OVX) and examined CUL4A expression. It was noted that CUL4A expression was increased in the bone marrow of OVX mice. Overexpression of CUL4A promoted osteoclast differentiation, and knockdown of CUL4A alleviated osteoporosis symptoms of OVX mice. Bioinformatic analyses were applied to identify the downstream target genes of microRNA‐340‐5p (miR‐340‐5p), followed by interaction analysis. The bone marrow macrophages (BMMs) were isolated from femur of OVX mice, which were transfected with different plasmids to alter the expression of CUL4A, Zinc finer E‐box binding homeobox 1 (ZEB1), miR‐340‐5p, and Toll‐like receptor 4 (TLR4). ChIP assay was performed to detect enrichment of ZEB1 promoter by H3K4me3 antibody in BMMs. ZEB1 was overexpressed in the bone marrow of OVX mice. Overexpression of CUL4A mediated H3K4me3 methylation to increase ZEB1 expression, thus promoting osteoclast differentiation. Meanwhile, ZEB1 could inhibit miR‐340‐5p expression and upregulate HMGB1 to induce osteoclast differentiation. Overexpressed ZEB1 activated the TLR4 pathway by regulating the miR‐340‐5p/HMGB1 axis to induce osteoclast differentiation, thus promoting the development of osteoporosis. Overall, E3 ubiquitin ligase CUL4A can upregulate ZEB1 to repress miR‐340‐5p expression, leading to HMGB1 upregulation and the TLR4 pathway activation, which promotes osteoclast differentiation and the development of osteoporosis. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is the most prevalent orthopedic malignancy with a dismal prognosis. The high iron absorption rate in OS cells of patients suggests that ferroptosis may be related to the progression of OS, but its potential molecular regulatory role is still unclear. Based on the ability to couple with exosomes for targeted delivery of signals, exosome-derived micro ribonucleic acids (miRNAs) can potentially serve as diagnostic biomarkers for OS. Methods: We identified ferroptosis-related miRNAs and messenger ribonucleic acids(mRNAs) in OS using bioinformatics analysis and performed survival analysis. Then we measured miRNA expression levels through exosome microarray sequencing, and used RT-qPCR and IHC to verify the expression level of miR-144-3p and ZEB1. Stable gene expression cell lines were fabricated for in vitro experiments. Cell viability, migration and invasion were determined by CCK-8 and transwell experiment. Use the corresponding reagent kit to detect GSH/GSSG ratio, Fe2+ level, MDA level and ROS level, and measure the expression levels of GPX4, ACSL4 and xCT through RT-qPCR and WB. We also constructed nude mice model for in vivo experiments. Finally, the stability of the miRNA/mRNA axis was verified through functional rescue experiments. Results: Low expression of miR-144-3p and high expression of ZEB1 in OS cell lines and tissues was observed. Overexpression of miR-144-3p can promote ferroptosis, reduce the survival ability of OS cells, and prevent the progression of OS. In addition, overexpression of miR-144-3p can downregulate the expression of ZEB1 in cell lines and nude mice. Knockdown of miR-144-3p has the opposite effect. The functional rescue experiment validated that miR-144-3p can regulate downstream ZEB1, and participates in the occurrence and development of OS by interfering with redox homeostasis and iron metabolism. Conclusions: MiR-144-3p can induce the occurrence of ferroptosis by negatively regulating the expression of ZEB1, thereby inhibiting the proliferation, migration, and invasion of OS cells. [ABSTRACT FROM AUTHOR]

Chemoresistance is associated with tumor recurrence, metastases, and short survival. Cisplatin is one of the most used chemotherapies in cancer treatment, including head and neck squamous cell carcinoma (HNSCC), and many patients develop resistance. Here, we established cell lines resistant to cisplatin to better understand epigenetics and biological differences driving the progression of HNSCC after treatment. Cisplatin resistance was established in CAL‐27 and SCC‐9 cell lines. Gene expression of HDAC1, HDAC2, SIRT1, MTA1, KAT2B, KAT6A, KAT6B, and BRD4 indicated the cisplatin activates the epigenetic machinery. Increases in tumor aggressiveness were detected by BMI‐1 and KI‐67 in more resistant cell lines. Changes in cellular shape and epithelial–mesenchymal transition (EMT) activation were also observed. HDAC1 and ZEB1 presented an opposite distribution with down‐regulation of HDAC1 and up‐regulation of ZEB1 in the course of chemoresistance. Up‐regulation of ZEB1 and BMI‐1 in patients with HNSCC is also associated with a poor response to therapy. Cancer stem cells (CSC) population increased significantly with chemoresistance. Down‐regulation of HDAC1, HDAC2, and SIRT1 and accumulation of Vimentin and ZEB1 were observed in the CSC population. Our results suggest that in the route to cisplatin chemoresistance, epigenetic modifications can be associated with EMT activation and CSC accumulation which originate more aggressive tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: The downstream signaling mechanisms and importance of the autocrine secretion of the vasoactive intestinal peptide (VIP) in cancer remains poorly understood. We hypothesized that VIP expression may promote cancer-associated signaling pathways. We analyzed gene sequencing data from cancer and healthy tissues based on the co-expression data of the VIP with 760 cancer-related genes. We identified a meaningful and novel association between the VIP and transcription factor ZEB1 in healthy and malignant human gastrointestinal tissues. ZEB1 is a known regulator of cancer EMT (epithelial–mesenchymal transition). Gene set analysis further supports the overlap in the EMT and cell cycle pathways. Our results identify a potentially novel function of the autocrine VIP as an important signaling peptide and biomarker of ZEB1-mediated EMT. VIP (vasoactive intestinal peptide) is a 28-amino acid peptide hormone expressed by cancer and the healthy nervous system, digestive tract, cardiovascular, and immune cell tissues. Many cancers express VIP and its surface receptors VPAC1 and VPAC2, but the role of autocrine VIP signaling in cancer as a targetable prognostic and predictive biomarker remains poorly understood. Therefore, we conducted an in silico gene expression analysis to study the mechanisms of autocrine VIP signaling in cancer. VIP expression from TCGA PANCAN tissue samples was analyzed against the expression levels of 760 cancer-associated genes. Of the 760 genes, 10 (MAPK3, ZEB1, TEK, NOS2, PTCH1 EIF4G1, GMPS, CDK2, RUVBL1, and TIMELESS) showed statistically meaningful associations with the VIP (Pearson's R-coefficient > |0.3|; p < 0.05) across all cancer histologies. The strongest association with the VIP was for the epithelial–mesenchymal transition regulator ZEB1 in gastrointestinal malignancies. Similar positive correlations between the VIP and ZEB1 expression were also observed in healthy gastrointestinal tissues. Gene set analysis indicates the VIP is involved in the EMT and cell cycle pathways, and a high VIP and ZEB1 expression is associated with higher median estimate and stromal scores These findings uncover novel mechanisms for VIP- signaling in cancer and specifically suggest a role for VIP as a biomarker of ZEB1-mediated EMT. Further studies are warranted to characterize the specific mechanism of this interaction. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is one of the most common and intractable microvascular complications of diabetes worldwide, serving as the main cause of terminal renal disease. Due to the lack of early specific symptoms and diagnostic markers, DN severely threatens the sufferer's life. MicroRNA-192 (miR-192) was early identified in human renal cortical tissue and stored and excreted in urine as microvesicles. MiR-192 was found to be involved in the development of DN. For the first time, the present review summarized all the current evidence on the topic of the roles of miR-192 in DN. Finally, 28 studies (ten clinical trials and eighteen experimental studies) were eligible for thorough reviewing. Most of the clinical trials (7/10, 70%) indicated miR-192 might be a protective factor for DN development and progression, while the majority of experimental studies (14/18, 78%) suggested miR-192 might be a pathogenic factor for DN. Mechanistically, miR-192 interacts with various direct targeted proteins (i.e., ZEB1, ZEB2, SIP1, GLP1R, and Egr1) and signaling cascades (i.e., SMAD/TGF-b and PTEN/PI3K/AKT), together contribute to the pathogenesis of DN through epithelial-to-mesenchymal transition (EMT), extracellular matrix deposition, and fibrosis formation. The current review highlights the dual role of miR-192 in the development of DN. Low serum miR-192 expression could be applied for the early prediction of DN (the early stage of DN), while the high miR-192 level in renal tissues and urine may imply the progression of DN (the late stage of DN). Further investigations are still warranted to illustrate this inconsistent phenomenon, which may facilitate promoting the therapeutic applications of miR-192 in predicting and treating DN. [ABSTRACT FROM AUTHOR]

P53 is a critical tumor suppressor that protects the integrity of genome and prevents cells from malignant transformation, including metastases. One of the driving forces behind the onset of metastases is the epithelial to mesenchymal transition (EMT) program. Zeb1 is one of the key transcription factors that govern EMT (TF-EMT). Therefore, the interaction and mutual influence of p53 and Zeb1 plays a critical role in carcinogenesis. Another important feature of tumors is their heterogeneity mediated by the presence of so-called cancer stem cells (CSCs). To this end, we have developed a novel fluorescent reporter-based approach to enrich the population of CSCs in MCF7 cells with inducible expression of Zeb1. Using these engineered cell lines, we studied the effect of p53 on Zeb1 interactomes isolated from both CSCs and regular cancer cells. By employing co-immunoprecipitations followed by mass spectrometry, we found that the composition of Zeb1 interactome was affected not only by the p53 status but also by the level of Oct4/Sox2 expression, indicating that stemness likely affects the specificity of Zeb1 interactions. This study, together with other proteomic studies of TF-EMT interactomes, provides a framework for future molecular analyses of biological functions of Zeb1 at all stages of oncogenesis. [ABSTRACT FROM AUTHOR]

Distal metastases from breast cancer, especially bone metastases, are extremely common in the late stages of the disease and are associated with a poor prognosis. EMT is a biomarker of the early process of bone metastasis, and MMP‐9 and MMP‐13 are important osteoclastic activators. Previously, we found that meso‐Hannokinol (HA) could significantly inhibit EMT and MMP‐9 and MMP‐13 expressions in breast cancer cells. On this basis, we further explored the role of HA in breast cancer bone metastasis. In vivo, we established a breast cancer bone metastasis model by intracardially injecting breast cancer cells. Intraperitoneal injections of HA significantly reduced breast cancer cell metastasis to the leg bone in mice and osteolytic lesions caused by breast cancer. In vitro, HA inhibited the migration and invasion of breast cancer cells and suppressed the expressions of EMT, MMP‐9, MMP‐13, and other osteoclastic activators. HA inhibited EMT and MMP‐9 by activating the ROS/JNK pathway as demonstrated by siJNK and SP600125 inhibition of JNK phosphorylation and NAC scavenging of ROS accumulation. Moreover, HA promoted bone formation and inhibited bone resorption in vitro. In conclusion, our findings suggest that HA may be an excellent candidate for treating breast cancer bone metastasis. [ABSTRACT FROM AUTHOR]

Ischemic stroke (IS) is the most common type of stroke and the second leading cause of death overall. Neural stem cells play protective roles in IS, but the underlying mechanism remains to be determined. Neural stem cells (NSC) were obtained from the fetal brain tissue of C57BL/6J mice. NSC-derived exosomes (NSC-Exos) were identified in the conditioned medium. Internalization of NSC-Exos was analyzed by fluorescence microscopy. In vitro microglia ischemic stroke injury model was induced using oxygen glucose deprivation/re-oxygenation (OGD/R) method. Cell viability and inflammation were analyzed by MTT, qPCR, ELISA and Western blotting assay. Interaction between ZEB1 and the promoter of GPR30 was verified by luciferase assay and chromatin immunoprecipitation. NSC-Exos prevented OGD/R-mediated inhibition of cell survival and the production of inflammatory cytokines in microglia cells. NSC-Exos increased ZEB1 expression in OGD/R-treated microglia. Down-regulation of ZEB1 expression in NSC-Exos abolished NSC-Exos' protective effects on OGD/R-treated microglia. ZEB1 bound to the promoter region of GPR30 and promoted its expression. Inhibiting GPR30 reversed NSC-Exos effects on cell viability and inflammation injury in OGD/R-treated microglia. Our study demonstrated that NSC exerted cytoprotective roles through release of exosomal ZEB1,which transcriptionally upregulated GPR30 expression, resulting in a reduction in TLR4/NF-κB pathway-induced inflammation. These findings shed light on NSC-Exos' cytoprotective mechanism and highlighted its potential application in the treatment of IS. [ABSTRACT FROM AUTHOR]

Background: Ultraviolet A (UVA) irradiation can lead to skin damage and premature skin aging known as photoaging. This work found that UVA irradiation caused an imbalance between dermal matrix synthesis and degradation through the aberrant upregulation of transgelin (TAGLN) and studied the underlying molecular mechanism. Results: Co-immunoprecipitation and proximal ligation assay results showed that TAGLN can interact with USP1. USP1 can be retained in the cytoplasm by TAGLN in UVA-induced cells, which inhibits the interaction between USP1/zinc finger E-box binding homeobox 1 (ZEB1), promote the ubiquitination degradation of ZEB1, and lead to photoaging. TAGLN knockdown can release USP1 retention and help human skin fibroblasts (HSFs) resist UVA-induced damage. The interactive interface inhibitors of TAGLN/USP1 were screened via virtual docking to search for small molecules that inhibit photoaging. Zerumbone (Zer), a natural product isolated from Zingiber zerumbet (L.) Smith, was screened out. Zer can competitively bind TAGLN to reduce the retention of USP1 in the cytoplasm and the degradation of ZEB1 ubiquitination in UV-induced HSFs. The poor solubility and permeability of Zer can be improved by preparing it as a nanoemulsion, which can effectively prevent skin photoaging caused by UVA in wild-type (WT) mice. Zer cannot effectively resist the photoaging caused by UVA in Tagln−/− mice because of target loss. Conclusions: The present results showed that the interaction of TAGLN and USP1 can promote ZEB1 ubiquitination degradation in UV-induced skin photoaging, and Zer can be used as an interactive interface inhibitor of TAGLN/USP1 to prevent photoaging. [ABSTRACT FROM AUTHOR]

Background: Globally, nasopharyngeal carcinoma (NPC) is a prevalent and deadly malignancy. Despite the role of methyltransferase like 13 (METTL13) having been highlighted in a majority of human cancers, its function and mechanism in NPC is indistinct. Methods: The expression level of METTL13 in NPC cell lines and normal cells was detected using a quantitative real‐time polymerase chain reaction. Gain‐ and loss‐of function experiments were conducted. Cell counting kit‐8, 5‐ethynyl‐2′‐deoxyuridine, wound‐healing, Transwell and tube formation assays, respectively, appraised the proliferative, migratory, invasive and angiogenic cellular responses. Corresponding protein expression was measured by western blotting. A chromatin immunoprecipitation assay was applied to verify the association between ZEB1 and the TPT1 promoter. Eventually, to substantiate the critical role of METTL13 in NPC, the establishment of an in vivo tumorigenesis model was accomplished. Results: METTL13 possessed fortified expression in NPC cells. METTL13 silencing markedly suppressed NPC cellular phenotypes in vitro, including proliferative, migratory, invasive and angiogenic events, as well as hindered tumorigenesis in vivo. Additionally, METTL13 positively regulated ZEB1, whereas ZEB1 could bind to TPT1 promoter and transcriptionally regulate TPT1. TPT1 was also found to be upregulated in NPC cells. TPT1 silencing suppressed NPC cellular phenotypes in vitro. TPT1 overexpression partly weakened the anti‐tumor effect of METTL13 in NPC. Conclusions: In summary, METTL13 up‐regulated ZEB1, which facilitated the transcriptional activation of TPT1, ultimately promoting NPC growth and metastasis, providing a potential therapeutic strategy for NPC treatment. [ABSTRACT FROM AUTHOR]

Background: Oral squamous cell carcinoma (OSCC), an HPV-negative head and neck cancer, frequently metastasizes to the regional lymph nodes but only occasionally beyond. Initial phases of metastasis are associated with an epithelial–mesenchymal transition (EMT), while the consolidation phase is associated with mesenchymal–epithelial transition (MET). This dynamic is referred to as epithelial–mesenchymal plasticity (EMP). While it is known that EMP is essential for cancer cell invasion and metastatic spread, less is known about the heterogeneity of EMP states and even less about the heterogeneity between primary and metastatic lesions. Methods: To assess both the heterogeneity of EMP states in OSCC cells and their effects on stromal cells, we performed single-cell RNA sequencing (scRNAseq) of 5 primary tumors, 9 matching metastatic and 5 tumor-free lymph nodes and re-analyzed publicly available scRNAseq data of 9 additional primary tumors. For examining the cell type composition, we performed bulk transcriptome sequencing. Protein expression of selected genes were confirmed by immunohistochemistry. Results: From the 23 OSCC lesions, the single cell transcriptomes of a total of 7263 carcinoma cells were available for in-depth analyses. We initially focused on one lesion to avoid confounding inter-patient heterogeneity and identified OSCC cells expressing genes characteristic of different epithelial and partial EMT stages. RNA velocity and the increase in inferred copy number variations indicated a progressive trajectory towards epithelial differentiation in this metastatic lesion, i.e., cells likely underwent MET. Extension to all samples revealed a less stringent but essentially similar pattern. Interestingly, MET cells show increased activity of the EMT-activator ZEB1. Immunohistochemistry confirmed that ZEB1 was co-expressed with the epithelial marker cornifin B in individual tumor cells. The lack of E-cadherin mRNA expression suggests this is a partial MET. Within the tumor microenvironment we found immunomodulating fibroblasts that were maintained in primary and metastatic OSCC. Conclusions: This study reveals that EMP enables different partial EMT and epithelial phenotypes of OSCC cells, which are endowed with capabilities essential for the different stages of the metastatic process, including maintenance of cellular integrity. During MET, ZEB1 appears to be functionally active, indicating a more complex role of ZEB1 than mere induction of EMT. [ABSTRACT FROM AUTHOR]

Osteoclasts are bone‐resorbing polykaryons responsible for skeletal remodeling during health and disease. Coincident with their differentiation from myeloid precursors, osteoclasts undergo extensive transcriptional and metabolic reprogramming in order to acquire the cellular machinery necessary to demineralize bone and digest its interwoven extracellular matrix. While attempting to identify new regulatory molecules critical to bone resorption, we discovered that murine and human osteoclast differentiation is accompanied by the expression of Zeb1, a zinc‐finger transcriptional repressor whose role in normal development is most frequently linked to the control of epithelial‐mesenchymal programs. However, following targeting, we find that Zeb1 serves as an unexpected regulator of osteoclast energy metabolism. In vivo, Zeb1‐null osteoclasts assume a hyperactivated state, markedly decreasing bone density due to excessive resorptive activity. Mechanistically, Zeb1 acts in a rheostat‐like fashion to modulate murine and human osteoclast activity by transcriptionally repressing an ATP‐buffering enzyme, mitochondrial creatine kinase 1 (MtCK1), thereby controlling the phosphocreatine energy shuttle and mitochondrial respiration. Together, these studies identify a novel Zeb1/MtCK1 axis that exerts metabolic control over bone resorption in vitro and in vivo. Synopsis: Osteoporosis and similar bone‐wasting conditions can be caused by an increase in osteoclast‐mediated bone resorption. Here, we learn that both murine and human osteoclasts are activated by a Zeb1‐dependent regulation of mitochondrial energy metabolism.The transcription factor Zeb1 is a negative regulator of giant multinucleated osteoclast‐mediated skeletal remodeling.Zeb1 regulates mitochondrial energy metabolism in myeloid lineage‐derived osteoclasts.The mitochondrial creatine kinase MtCK1 is a transcriptional target of Zeb1 that enables bone resorption by buffering ATP availability. [ABSTRACT FROM AUTHOR]

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann–Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology. [ABSTRACT FROM AUTHOR]

Corneal dystrophies (CDs) represent a group of inherited diseases characterized by the progressive deposit of abnormal materials in the cornea. This study aimed to describe the variant landscape of 15 genes responsible for CDs based on a cohort of Chinese families and a comparative analysis of literature reports. Families with CDs were recruited from our eye clinic. Their genomic DNA was analyzed using exome sequencing. The detected variants were filtered using multi-step bioinformatics and confirmed using Sanger sequencing. Previously reported variants in the literature were summarized and evaluated based on the gnomAD database and in-house exome data. In 30 of 37 families with CDs, 17 pathogenic or likely pathogenic variants were detected in 4 of the 15 genes, including TGFBI, CHST6, SLC4A11, and ZEB1. A comparative analysis of large datasets revealed that 12 of the 586 reported variants are unlikely causative of CDs in monogenic mode, accounting for 61 of 2933 families in the literature. Of the 15 genes, the gene most frequently implicated in CDs was TGFBI (1823/2902, 62.82% of families), followed by CHST6 (483/2902, 16.64%) and SLC4A11 (201/2902, 6.93%). This study presents, for the first time, the landscape of pathogenic and likely pathogenic variants in the 15 genes responsible for CDs. Awareness of frequently misinterpreted variants, such as c.1501C>A, p.(Pro501Thr) in TGFBI, is crucial in the era of genomic medicine. [ABSTRACT FROM AUTHOR]

Medulloblastoma (MB) is a malignant brain tumor that afflicts mostly children and adolescents and presents four distinct molecular subgroups, known as WNT, SHH, Group 3, and Group 4. ZEB1 is a transcription factor that promotes the expression of mesenchymal markers while restraining expression of epithelial and polarity genes. Because of ZEB1 involvement in cerebellum development, here we investigated the role of ZEB1 in MB. We found increased expression of ZEB1 in MB tumor samples compared to normal cerebellar tissue. Expression was higher in the SHH subgroup when compared to all other MB molecular subgroups. High ZEB1 expression was associated with poor prognosis in Group 3 and Group 4, whereas in patients with WNT tumors poorer prognosis were related to lower ZEB1 expression. There was a moderate correlation between ZEB1 and MYC expression in Group 3 and Group 4 MB. Treatment with the immunomodulator and histone deacetylase (HDAC) inhibitor fingolimod (FTY720) reduced ZEB1 expression specifically in D283 cells, which are representative of Group 3 and Group 4 MB. These findings reveal novel subgroup-specific associations of ZEB1 expression with survival in patients with MB and suggest that ZEB1 expression can be reduced by pharmacological agents that target HDAC activity. [ABSTRACT FROM AUTHOR]

Simple Summary: Mycosis fungoides (MF) is the most common variety of cutaneous T-cell lymphoma. Our previous studies showed that the epithelial–mesenchymal transition (EMT) transcription factors (TFs) Twist1 and Zeb1 have prognostic value in MF. The main objective of the present study was to gain better knowledge about the biological mechanisms behind this phenomenon. The RNA of 40 skin tumor biopsies (from 40 patients) was sequenced and analyzed. Twist1 protein expression seemed to classify MF cases into different groups based on their global RNA expression. Additionally, high Twist1 protein expression was associated with several genes and pathways known to have roles in aggressive tumor biology. For Zeb1, similar results were not found. Our results suggest Twist1 to be a central transcription factor and pathway regulator in the disease progression of MF. Twist1 might be an interesting object for developing targeted therapies for MF. The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I–IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF. [ABSTRACT FROM AUTHOR]

Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAMdownstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDAMutZEB1), that stably express MutZEB1 (DZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (DZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. Conclusions: These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells. [ABSTRACT FROM AUTHOR]

Cancer-associated fibroblasts (CAFs) are a prominent component in the tumor microenvironment (TME), which plays an important role in lung carcinogenesis. Here, we investigated microenvironmental markers expressed by CAFs, including α-smooth muscle actin, CD10, podoplanin, fibroblast-specific protein 1, platelet-derived growth factor α and β, fibroblast-associated protein, tenascin-C, zinc finger E-box binding homeobox 1 (ZEB1), and twist-related protein 1 expression levels. We evaluated samples from 257 patients with lung adenocarcinoma (LAD) to assess the associations of CAF-related protein expression patterns with prognosis. LAD cases were stratified using cluster analysis. To determine the utility of prognostic markers in LAD, univariate and multivariate analyses were performed. LAD cases were classified into subgroups 1 and 2. Subgroup 2 was shown to be significantly correlated with disease-free and overall survival using univariate and multivariate analyses in this group. Upregulation of podoplanin was identified as a single prognostic marker in this study by univariate and multivariate analyses. In addition, ZEB1 overexpression was correlated with disease-free survival. Our current results suggested that the specific CAF phenotype (e.g., the expression pattern of CAF-related proteins) could predict outcomes in patients with LAD. In addition, podoplanin upregulation may predict outcomes in these patients. [ABSTRACT FROM AUTHOR]

Simple Summary: While invading tumor-adjacent tissue, cancer cells often undergo epithelial-mesenchymal transition (EMT). A mechanistic understanding of EMT may therefore improve diagnostic and therapeutic options. Traditionally, EMT is thought to be regulated by SNAIL, TWIST, and ZEB transcription factors. Since this view is increasingly challenged, we aimed to examine the importance of traditional EMT regulators while identifying alternative factors potentially orchestrating EMT. By combining computational analyses of epigenomic and transcriptomic data with loss-of-function experiments, we demonstrate that JUNB, a component of the AP-1 transcription factor complex, is crucial for EMT in the MCF10A mammary epithelial cell line model, but not SNAIL proteins and ZEB1. We exploited the JUNB-dependence of EMT to define a gene signature which is controlled by TGFβ in multiple cancer entities and which is predictive of patient survival. Our results provide a more refined picture of the dynamic regulation of EMT and suggest that traditional models for EMT regulation may need to be revised. Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options. [ABSTRACT FROM AUTHOR]

Posterior polymorphous corneal dystrophy (PPCD), a rare, bilateral, autosomal-dominant, inherited corneal dystrophy, affects the Descemet membrane and corneal endothelium. We describe an unusual presentation of PPCD associated with a previously unknown genetic alteration in the ZEB1 gene. The proband is a 64-year-old woman diagnosed with keratoconus referred for a corneal endothelium study who presented endothelial lesions in both eyes suggestive of PPCD, corectopia and iridocorneal endothelial synechiae in the right eye and intrastromal segments in the left eye. The endothelial count was 825 in the right eye and 1361 in the left eye, with typical PPCD lesions visible under specular and confocal microscopy. In the next generation sequencing genetic analysis, a heterozygous c.1A > C (p.Met1Leu) mutation was found in the ZEB1 gene (TCF8). The PPCD3 subtype is associated with corneal ectasia, and both can appear due to a pathogenic mutation in the ZEB1 gene (OMIM #189909). However, our patient had a previously unreported mutation in the ZEB1 gene, which mediates the transition between cell lines and provides a pathogenic explanation for the epithelialisation of the corneal endothelium, a characteristic of PPCD. [ABSTRACT FROM AUTHOR]

Background: Invasion and metastasis in tumors are considered as two most important reasons for the reduction of treatment efficiency, as well as an increase in the mortality rate among the patients. According to the evidence, HOX transcript antisense RNA (HOTAIR) accounts for one of the main IncRNAs associated with the development and expansion of gastric cancer (GC). Objectives: This research investigates the impact of HOTAIR suppression on the expression and translation of the ZEB1 marker in GC. Methods: The knockdown HOTAIR was in the AGS cell line using small interfering RNA targeting against HOTAIR (si-HOTAIR). The impact of HOTAIR expression on the cell proliferation was previously evaluated by the MTT assay. The expression of the zinc finger Ebox binding homeobox 1 (ZEB1) gene in transfected cell lines was quantified compared to the control samples using the quantitative real-time polymerase chain reaction (qRT-PCR). The enzyme-linked immunosorbent assay (ELISA) was also used to assess the HOTAIR suppression impact on the ZEB1 protein. Results: The findings revealed that a decrease in the HOTAIR expression could cause a reduction in the proliferation and growth of cancer cells (Fold changes = 0/28, P-value < 0.01). According to the impact of changes in the HOTAIR expression levels on the ZEB1 expression, the ZEB1 expression level was directly correlated with HOTAIR so that a decrease in the HOTAIR expression led to a significant decline in the ZEB1 expression level (P-value< 0.05). At the protein level, the effect of theknockdownof HOTAIR expression on the reduction of ZEB1 protein was also observed. Conclusions: Our findings showed a significant association between the HOTAIR and ZEB1 expression levels. Overall, the HOTAIRZEB1 axis plays a vital role in the epithelial-to-mesenchymal transition (EMT) process in human GC and represents a new therapeutic strategy for future GC treatment. [ABSTRACT FROM AUTHOR]

Background: Epithelial-mesenchymal transition (EMT) plays an important role in bladder carcinoma (BC) invasiveness and metastasis. Studies have shown that muscle-invasive BC (MIBC) and non-MIBC (NMIBC) are different at the molecular level owing to different EMT-related programming. Recent studies suggest that dysregulation of specific miRNAs is linked to EMT in BC. With this background, we aimed to study the immunoexpression of EMT-markers and its correlation with miRNA-200c expression in a series of MIBCs and NMIBCs. Materials and Methods: Quantitative real-time-polymerase chain reaction for the quantification of miR-200c expression was performed on 50 cases of urinary BC obtained from transurethral resection of bladder tumor (TURBT), cystectomy specimens, and ten peritumoral bladder tissue. Immunohistochemistry for ZEB1, ZEB2, TWIST, E-cadherin, and β-catenin was performed on tumor and peritumoral bladder tissue. Results: Thirty-five TURBT and 15 cystectomy specimens were assessed. Among MIBC, loss of expression of E-cadherin (72.3%), β-catenin (66.7%), and ZEB1, ZEB2, and TWIST2 immunoreactivity was noted in 53.3%, 86.7%, and 73.3% of cases, respectively. Among NMIBC, loss of expression of E-cadherin (22.5%), β-catenin (17.1%) and ZEB1, ZEB2, and TWIST immunoreactivity was noted in 11.5%, 51.4%, and 91.4% of cases, respectively. Upregulation of miRNA-200c was noted in cases with retained E-cadherin and negative TWIST expression. Downregulation of miRNA-200c expression was noted in all the cases showing loss of E-cadherin, β-catenin, and in cases immunoreactive for ZEB1, ZEB2, and TWIST in MIBC. Downregulation of miRNA-200c expression was also noted in cases of MIBC with retained β-catenin and those immunonegative for ZEB1 and ZEB2. A similar trend was noted in NMIBC. Median miRNA-200c expression was low in both high-grade and low-grade NMIBC compared to peritumoral bladder tissue and was not statistically significant. Conclusion: This study for the first time explores the relation of miR200C with E-cadherin, b-catenin, and its direct transcriptional regulators, namely Zeb1, Zeb2, and Twist in the same cohort of BC. We observed that miRNA-200c is downregulated in both MIBC and NMIBC. We identified novel expression of TWIST in cases of BC showing downregulation of miR200Cs suggesting that it is one of the protein targets of altered miRNA-200c expression contributing to EMT and can serve as a promising diagnostic marker and therapeutic target. Loss of E-cadherin and ZEB1 immunoexpression in high-grade NMIBC suggests an aggressive clinical behavior. However, ZEB2 heterogeneous expression in BC limits its diagnostic and prognostic utility. [ABSTRACT FROM AUTHOR]