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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]

Background: Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. Methods: Kaplan–Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. Results: In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferase reporter assay verified that LAMC2 targeted ZEB1 to promote its transcription. Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. Conclusion: LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients. [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: 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]

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]

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]

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]

Background: Breast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain. Methods: We identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP. Results: PAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3'UTR region, respectively. Conclusion: In summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy. [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: 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]

Background: Long intergenic non-coding RNA 00963 (LINC00963) is an oncogenic lncRNA in human cancers. However, little is known on how it impacts the pathogenesis of lung adenocarcinoma (LUAD). Methods: Biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) were examined by CCK-8, colony formation, EdU incorporation, transwell, and immunofluorescence assays, respectively. Macrophage polarization was evaluated by flow cytometry. Ubiquitination of Zeb1 was examined by co-immunoprecipitation. The location of LINC00963 in LUAD tissues and cell lines was tested by FISH assay. The LINC00963/HNRNPA2B1/Siah1 mRNA complex interaction was verified using RNA pull-down and immunoprecipitation assays. The exact roles of LINC00963 were further validated in metastasis and xenograft models. Results: Higher LINC00963 expression in LUAD patients positively correlated with shorter overall survival, higher stages, and metastasis. LINC00963 mainly localized in the cytoplasm and aggravated malignant phenotypes of LUAD cells in vitro and metastasis in vivo. Mechanistically, LINC00963 directly interacted HNRNPA2B1 protein to trigger the degradation of Siah1 mRNA, which inhibited the ubiquitination and degradation of Zeb1. Moreover, exosomal LINC00963 derived from LUAD cells induced M2 macrophage polarization and promoted LUAD growth and metastasis. Conclusion: By stabilizing Zeb1 in cancer cells and delivering exosomes to induce M2 macrophage polarization, LINC00963 promoted the malignancy and metastasis of LUAD. Targeting LINC00963 may become a valuable therapeutic target for LUAD. [ABSTRACT FROM AUTHOR]

Background: DNMT1 has been shown to be highly expressed in a variety of cancers, including breast cancer. However, the mechanism is not very clear. Therefore, we aim to reveal the mechanism of DNMT1 highly express in breast cancer. And we also want to explore the role of DNMT1 in tumour microenvironment promoting breast cancer progression. Results: In this study, we demonstrate that DNMT1 is overexpressed in breast cancer and that DNMT1 promotes breast cancer tumorigenesis and metastasis. We discovered that ZEB1 activates DNMT1 expression in breast cancer cells by recruiting P300 binding to the DNMT1 promoter and increasing its acetylation. Moreover, we revealed that tumour-associated macrophages (TAMs) increase DNMT1 expression in breast cancer cells via the IL-6-pSTAT3-ZEB1-DNMT1 axis in the tumour microenvironment. DNMT1 is required for TAM-mediated breast cancer cell migration. In addition, we confirmed that there were positive correlations among CD163 (TAM marker) expression, ZEB1 expression and DNMT1 expression in breast cancer patient tissues. Conclusions: Our study indicates that DNMT1 is necessary for TAM-mediated breast cancer metastasis. Decitabine (DAC), as a specific DNA methylation inhibitor and FDA-approved drug, is a bona fide drug for breast cancer treatment. [ABSTRACT FROM AUTHOR]

Background: Osteoporosis (OP) is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the role of zinc finger E-box binding homeobox 1 (ZEB1) in OP. Methods: First, gene expression microarrays for OP were downloaded from the Gene Expression Omnibus database and analyzed to screen for potential targets. Subsequently, a rat OP model was constructed using ovariectomy (OVX), and osteoblastic and osteoclastic differentiation and alterations in osteoporotic symptoms were observed upon intraperitoneal injection of oe-ZEB1 lentiviral vectors. DNA polymerase delta interacting protein 2 (POLDIP2) was predicted to be a downstream target of ZEB1, which was validated by ChIP-qPCR and dual-luciferase experiments. RAW264.7 cells were subjected to lentiviral vector infection of oe-ZEB1 and/or sh-POLDIP2, followed by RANKL treatment to induce osteoclast differentiation. Results: ZEB1 was poorly expressed in blood samples of postmenopausal patients with OP and in bone tissues of OVX-treated rats. Overexpression of ZEB1 or POLDIP2 in OVX rats promoted osteoblastogenesis and inhibited osteoclast differentiation. In RANKL-treated RAW264.7 cells, the transcription factor ZEB1 enhanced the expression of POLDIP2, and silencing of POLDIP2 attenuated the inhibitory effect of oe-ZEB1 on the differentiation of macrophages RAW264.7 to osteoclasts. Conclusions: ZEB1 promotes osteoblastogenesis and represses osteoclast differentiation, ultimately reducing the occurrence of postmenopausal OP by elevating the expression of POLDIP2. [ABSTRACT FROM AUTHOR]

Background: Breast cancer has become the most frequently diagnosed cancer worldwide. Increasing evidence indicated that zinc finger proteins (ZNFs), the largest family of transcription factors, contribute to cancer development and progression. Although ZNF384 is overexpressed in several types of human cancer, the role of ZNF384 in breast cancer remains unknown. Therefore, our research focused on ZNF384 regulation of the malignant phenotype of breast cancer and the underlying molecular mechanisms. Methods: CCK-8 and colony formation assays were used to evaluate cell proliferation. Transwell and scratch assays were used to evaluate the cell migration and invasion. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter assays were used to confirm the target relationship between ZNF384 and zinc finger E-box binding homeobox 1 (ZEB1). Xenografts were used to monitor the targets in vivo effects. Results: We noted that ZNF384 was significantly overexpressed in breast cancer and highlighted the oncogenic mechanism of ZNF384. ZNF384 transactivated ZEB1 expression and induced an epithelial and mesenchymal-like phenotype, resulting in breast cancer metastasis. Furthermore, ZNF384 may be a target of miR-485-5p, and ZEB1 can up-regulate ZNF384 expression by repressing miR-485-5p expression. Together, we unveiled a feedback loop of ZNF384–ZEB1 in breast cancer metastasis. Conclusions: The findings suggest that ZNF384 can serve as a prognostic factor and a therapeutic target for breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: Epithelial-to-mesenchymal transition (EMT) encompasses a highly dynamic and complex key process which leads to metastatic progression. In high-grade serous ovarian carcinoma (HG-SOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling promotes EMT driving tumor progression. However, the complex nature of intertwined regulatory circuits activated by ET-1 to trigger the metastatic process is not fully elucidated. Methods: The capacity of ET-1 pathway to guide a critical transcriptional network that is instrumental for metastatic growth was identified in patient-derived HG-SOC cells and cell lines through immunoblotting, q-RT-PCR, co-immunoprecipitation, in situ proximity ligation, luciferase reporter, chromatin immunoprecipitation assays and publicly available databases. Functional assays in HG-SOC cells and HG-SOC xenografts served to test the inhibitory effects of ET-1 receptors (ET-1R) antagonist in vitro and in vivo. Results: We demonstrated that ET-1/ETAR axis promoted the direct physical ZEB1/YAP interaction by inducing their nuclear accumulation in HG-SOC cells. Moreover, ET-1 directed their engagement in a functional transcriptional complex with the potent oncogenic AP-1 factor JUN. This led to the aberrant activation of common target genes, including EDN1 (ET-1) gene, thereby creating a feed-forward loop that sustained a persistent ET-1/ZEB1 signaling activity. Notably, ET-1-induced Integrin-linked kinase (ILK) signaling mediated the activation of YAP/ZEB1 circuit driving cellular plasticity, invasion and EMT. Of therapeutic interest, treatment of HG-SOC cells with the FDA approved ET-1R antagonist macitentan, targeting YAP and ZEB1-driven signaling, suppressed metastasis in vivo in mice. High gene expression of ETAR/ILK/YAP/AP-1/ZEB1 was a strong predictor of poor clinical outcome in serous ovarian cancer patients, indicating the translational relevance of this signature expression. Conclusions: This study provides novel mechanistic insights of the ET-1R-driven mediators that support the ability of HG-SOC to acquire metastatic traits which include the cooperation of YAP and ZEB1 regulatory circuit paving the way for innovative treatment of metastatic ovarian cancer. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is a common malignancy. Long non-coding RNAs (lncRNAs) partake in the progression of HCC. However, the role of lncRNA MAPKAPK5-AS1 in the development of HCC has not been fully clarified. Methods: RNA sequencing data and quantitative real-time polymerase chain reaction (qRT-PCR) were adopted to analyze MAPKAPK5-AS1, miR-429 and ZEB1 mRNA expressions in HCC tissues and cell lines. Western blot was used to detect ZEB1, E-cadherin and N-cadherin protein expressions. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Transwell and flow cytometry assays were adopted to analyze the effects of MAPKAPK5-AS1 on cell proliferation, migration, invasion and apoptosis. Besides, luciferase reporter assay was used to detect the targeting relationship between miR-429 and MAPKAPK5-AS1 or ZEB1 3'UTR. The xenograft tumor mouse models were used to explore the effect of MAPKAPK5-AS1 on lung metastasis of HCC cells. Results: MAPKAPK5-AS1 and ZEB1 expressions were up-regulated in HCC tissues, and miR-429 expression is down-regulated in HCC tissues. MAPKAPK5-AS1 knockdown could significantly impede HCC cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), as well as promote cell apoptosis. MAPKAPK5-AS1 overexpression could enhance L02 cell proliferation, migration, invasion and EMT, and inhibit cell apoptosis. MiR-429 was validated to be the target of MAPKAPK5-AS1, and miR-429 inhibitors could partially offset the effects of knocking down MAPKAPK5-AS1 on HCC cells. MAPKAPK5-AS1 could positively regulate ZEB1 expression through repressing miR-429. Moreover, fewer lung metastatic nodules were observed in the lung tissues of nude mice when the MAPKAPK5-AS1 was knocked down in HCC cells. Conclusion: MAPKAPK5-AS1 can adsorb miR-429 to promote ZEB1 expression to participate in the development of HCC. [ABSTRACT FROM AUTHOR]

Background: Emerging evidence has indicated the critical role of TRPV4 in diverse human cancers. However, the underlying molecular mechanism of TRPV4 in colon cancer invasiveness is still unknown.Methods: Immunohistochemistry staining was used to analyze the expression of TRPV4 and ZEB1 in clinical tissues; Wound healing and transwell assays were applied to determine the cell invasiveness; Western blot was used to explore the relation between TRPV4 and ZEB1.Results: Colon cancer cells were transfected with siRNA against TRPV4 or HC067047 (a selective TRPV4 antagonist), TRPV4 full-length plasmid or siRNA against ZEB1, or both, in order to measure cell migration and invasion. And we found that TRPV4 silencing or inhibition exhibited an inhibitory role in colon cancer cell migration and invasion, coupled with compromised EMT process, and suppressed AKT activity. TRPV4 stimulated expression of ZEB1 and consequently contributed to EMT process and invasiveness. It was also revealed that overexpression of TRPV4 and ZEB1 in clinical patients with local metastasis, and positive correlation between TRPV4 and ZEB1.Conclusions: Our results uncovered the role of TRPV4 in tumor metastasis and highlighted the potential mechanism of TRPV4-ZEB1 axis in indicating EMT. [ABSTRACT FROM AUTHOR]

Background: Although thousands of long noncoding RNAs (lncRNAs) have been annotated, only a few lncRNAs have been characterized functionally. In this study, we aimed to identify novel lncRNAs involved in the progression of gastric carcinoma (GC) and explore their regulatory mechanisms and clinical significance in GC. Methods: A lncRNA expression microarray was used to identify differential lncRNA expression profiles between paired GCs and adjacent normal mucosal tissues. Using the above method, the lncRNA SGO1-AS1 was selected for further study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) were performed to detect SGO1-AS1 expression in GC tissues. Gain-of-function and loss-of-function analyses were performed to investigate the functions of SGO1-AS1 and its upstream and downstream regulatory mechanisms in vitro and in vivo. Results: SGO1-AS1 was downregulated in gastric carcinoma tissues compared to adjacent normal tissues, and its downregulation was positively correlated with advanced clinical stage, metastasis status and poor patient prognosis. The functional experiments revealed that SGO1-AS1 inhibited GC cell invasion and metastasis in vitro and in vivo. Mechanistically, SGO1-AS1 facilitated TGFB1/2 mRNA decay by competitively binding the PTBP1 protein, resulting in reduced TGFβ production and, thus, preventing the epithelial-to-mesenchymal transition (EMT) and metastasis. In addition, in turn, TGFβ inhibited SGO1-AS1 transcription by inducing ZEB1. Thus, SGO1-AS1 and TGFβ form a double-negative feedback loop via ZEB1 to regulate the EMT and metastasis. Conclusions: SGO1-AS1 functions as an endogenous inhibitor of the TGFβ pathway and suppresses gastric carcinoma metastasis, indicating a novel potential target for GC treatment. [ABSTRACT FROM AUTHOR]

Aims: We focused on the detailed functions of circ-ABCB10 in cervical cancer (CC) development and its mechanisms. Background: The increasing findings have proposed the central roles of circular RNAs (circRNAs) in the tumorigenesis of various human cancers. Circ-ABCB10 displays promising oncogenic effect in several tumors. Methods: Circ-ABCB10 and miR-128-3p production levels in CC tissues and cells were tested through RT-qPCR. The association of circ-ABCB10 expression with clinicopathologic parameters of CC patients was statistically analyzed. Cell proliferation, invasion, apoptosis, and epithelial-mesenchymal transition (EMT) were evaluated by MTT, transwell invasion assays, flow cytometry analyses, and western blot examination of EMT markers. The binding activity between miR-128-3p and circ-ABCB10 or zinc finger E-box binding homeobox 1 (ZEB1) was explored through pull-down assay or luciferase reporter assay. The influence of circ-ABCB10 on CC tumorigenesis was evaluated by in vivo xenograft experiments. Results: The elevated circ-ABCB10 expression was determined in CC tissues and cells. Moreover, higher production level of circ-ABCB10 was close related to lymph-node metastasis, Federation of Gynecology and Obstetrics (FIGO) stage, and tumor size in CC patients. Loss of circ-ABCB10 weakened cell proliferative and invasive abilities, inhibited EMT, and induced apoptosis in CC. Loss of circ-ABCB10 inhibited ZEB1 expression by serving as a sponge of miR-128-3p in CC cells. Circ-ABCB10 sponged miR-128-3p to enhance cell proliferation, invasion, EMT and inhibit apoptosis in CC cells. Xenograft tumor assays confirmed that circ-ABCB10 knockdown inhibited CC tumor growth. Conclusion: Our study suggests that circ-ABCB10 depletion inhibits proliferation, invasion and EMT and promotes apoptosis of cervical cancer cells through miR-128-3p/ZEB1 axis and represses CC tumor growth. [ABSTRACT FROM AUTHOR]

Background: Metastasis is a major challenge in cervical cancer treatment. Previous studies have shown that the dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) promotes tumor metastasis and is overexpressed in cervical cancer. However, the biological role and mechanism of APE1 in cervical cancer metastasis have rarely been studied. Methods: We used gene set enrichment analysis (GSEA) to determine the APE1-related signaling pathways in cervical cancer. To investigate the role and mechanism of APE1 in cervical cancer metastasis and invasion, immunohistochemistry, immunofluorescence, western blotting, secondary structure prediction, coimmunoprecipitation, luciferase reporter, and electrophoretic mobility shift assays were performed. The inhibitory effects of the APE1 redox function inhibitor APX3330 on cervical cancer metastasis were evaluated using animal models. Results: Clinical data showed that high expression of APE1 was associated with lymph node metastasis in cervical cancer patients. GSEA results showed that APE1 was associated with epithelial to mesenchymal transition (EMT) in cervical cancer. Ectopic expression of APE1 promoted EMT and invasion of cervical cancer cells, whereas inhibition of APE1 suppressed EMT and invasion of cervical cancer cells in a redox function-dependent manner. Notably, APE1 redox function inhibitor APX3330 treatment dramatically suppressed cervical cancer cell lymph node and distant metastasis in vivo. Furthermore, we found that APE1 enhanced the interaction between ZEB1 and the E-cadherin promoter by binding to ZEB1, thereby suppressing the expression of E-cadherin, a negative regulator of EMT. Conclusion: Our findings help to elucidate the role played by APE1 in cervical cancer metastasis and targeting APE1 redox function may be a novel strategy for inhibiting cervical cancer metastasis. [ABSTRACT FROM AUTHOR]

Background: Invasive bladder tumors cause a worse prognosis in patients and remain a clinical challenge. Epithelial–mesenchymal transition (EMT) is associated with bladder cancer metastasis. In the present research, we attempted to demonstrate a novel mechanism by which a long noncoding RNA (lncRNA)-miRNA-mRNA axis regulates EMT and metastasis in bladder cancer. Methods: Immunofluorescence (IF) staining was used to detect Vimentin expression. The protein expression of ZEB1, Vimentin, E-cadherin, and Snail was investigated by using immunoblotting assays. Transwell assays were performed to detect the invasive capacity of bladder cancer cells. A wound healing assay was used to measure the migratory capacity of bladder cancer cells. Results: Herein, we identified lncRNA VIM-AS1 as a highly- expressed lncRNA in bladder cancer, especially in metastatic bladder cancer tissues and high-metastatic bladder cancer cell lines. By acting as a ceRNA for miR-655, VIM-AS1 competed with ZEB1 for miR-655 binding, therefore eliminating the miR-655-mediated suppression of ZEB1, finally promoting EMT in both high- and low-metastatic bladder cancer cells and enhancing cancer cell metastasis. Conclusions: In conclusion, the VIM-AS1/miR-655/ZEB1 axis might be a promising target for improving bladder cancer metastasis via an EMT-related mechanism. [ABSTRACT FROM AUTHOR]

Background: Breast cancer (BC) is a common malignancy worldwide. It has been reported that long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is abnormally expressed in BC. However, the role of HOTAIR in the malignancy of BC is worth further discussion. This study aims to clarify the function and molecular mechanism of HOTAIR in BC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expression of HOTAIR, microRNA (miR)-601 and zinc finger E-box binding homeobox 1 (ZEB1). Cell counting kit-8 (CCK-8) and transwell assay were used to detect the proliferation, migration and invasion of cells. Further, the protein levels of AKT, phosphorylated-AKT (p-AKT), ZEB1 and Ki-67 were confirmed by western blot (WB) assay. Moreover, dual-luciferase reporter assay was applied to examine the targeting relationship between HOTAIR and miR-601 or miR-601 and ZEB1. In addition, animal experiments were conducted to verify the effect of HOTAIR on BC tumor growth in vivo. Results: HOTAIR was upregulated in BC tissues and cells, and its knockdown suppressed the proliferation, migration, invasion and the activity of AKT signaling pathway of BC cells. HOTAIR could serve as a sponge of miR-601. Further experiments revealed that miR-601 inhibitor could reverse the inhibition effect of HOTAIR silencing on the progression of BC. Meanwhile, ZEB1 was a target of miR-601, and its overexpression could invert the suppression effect of miR-601 overexpression on the progression of BC. Additionally, ZEB1 expression was regulated by HOTAIR and miR-601. Furthermore, interference of HOTAIR could attenuate BC tumor growth in vivo. Conclusion: In short, this study demonstrated that HOTAIR promoted the proliferation, migration, invasion of BC through regulating the miR-601/ZEB1 axis, which provided a theoretical basis for the research on lncRNA-directed therapeutics in BC. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) have been demonstrated to exert crucial mediators in tumor initiation and development. Nevertheless, the roles of circKIF4A in breast cancer (BC) are still not very clear. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circKIF4A, miR-152, zinc finger E-box binding homeobox 1 (ZEB1) mRNA and caspase-3. Western blot assay was utilized to examine the protein level of ZEB1. Transwell assay and flow-cytometric analysis were adopted for the evaluation of cell migration, invasion and apoptosis, respectively. The associations among circKIF4A, miR-152 and ZEB1 were predicted by online websites and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Results: CircKIF4A and ZEB1 were conspicuously upregulated and miR-152 was markedly reduced in BC tissues and cells. Deficiency of circKIF4A repressed migration, invasion and induced apoptosis of BC cells. Moreover, circKIF4A was confirmed to be a sponge of miR-152 and miR-152 could bind to ZEB1. MiR-152 inhibition or ZEB1 overexpression abolished the impacts of circKIF4A knockdown on cell migration, invasion and apoptosis in BC. Conclusion: Silencing of circKIF4A hampered cell metastasis and promoted apoptosis by regulating ZEB1 via sponging miR-152 in BC. [ABSTRACT FROM AUTHOR]

Background: Our previous study showed Musashi2 (MSI2) promoted chemotherapy resistance and pernicious biology of pancreatic cancer (PC) by down-regulating Numb and p53. We further explored the novel molecular mechanism involving its oncogenic role in PC development. Methods: We investigated the potential role and mechanism of MSI2 in EGF-induced EMT in PC in vitro and vivo. Results: EGF enhanced EGFR (epidermal growth factor receptor) phosphorylation, induced EMT and activated ZEB1-ERK/MAPK signaling in 2 PC cells. However, MSI2 silencing reversed EGF stimulated function, including inhibiting EGF-promoted EMT-like cell morphology and EGF-enhanced cell invasion and migration. Meanwhile, MSI2 silencing inhibited EGF-enhanced EGFR phosphorylation at tyrosine 1068 and reversed EGF-induced change of the key proteins in EMT and ZEB1-ERK/MAPK signaling (ZEB1, E-cad, ZO-1, β-catenin, pERK and c-Myc). Additionally, MSI2 was co-stained and co-immunoprecipitated with ZEB1, pERK and c-Myc in PC cells by IF and co-IP, implying a close interaction between them. In vivo, MSI2 silencing inhibited pancreatic tumor size in situ and distant liver metastases. A close relationship of MSI2 with EMT and ZEB1-ERK/MAPK signaling were also observed in vivo and human PC samples, which coordinately promoted the poor prognosis of PC patients. Conclusions: MSI2 promotes EGF-induced EMT in PC via ZEB1-ERK/MAPK signaling. [ABSTRACT FROM AUTHOR]

Background: It was demonstrated that long non-coding RNAs occupied an important position in tumor pathogenesis and progression. We have previously found that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) promotes cell proliferation and metastases in pancreatic ductal adenocarcinoma (PDAC). The present study was aimed to discuss the underlying mechanisms.Methods: Bioinformatics method was used to identify the miRNA target of MALAT-1. Expressions of relative genes were assessed by quantitative real-time PCR and western blotting, respectively. Sulforhodamine B assay and Transwell assay were employed to detect cell proliferation, migration and invasion, respectively. Moreover, RNA immunoprecipitation was performed to determine whether RNA-induced silencing complex contained MALAT-1 and its potential binding miRNA. Luciferase assays was used to confirm potential binding site.Results: Bioinformatics search predicted that miR-200c-3p was a direct target of MALAT-1. Further, we found a reciprocal suppression between MALAT-1 and miR-200c-3p expression. In terms of mechanisms, high MALAT-1 and low miR-200c-3p may form a novel feedback loop. On the one hand, MALAT-1 functioned as a competing endogenous RNA to suppress miR-200c-3p expression, leading to upregulation of ZEB1 expression. On the other hand, miR-200c-3p inhibited the level of MALAT-1 expression was in a way similar to miRNA-mediated downregulation of target genes. Clinical data further indicated that MALAT-1 and ZEB1 expression was negatively correlated with miR-200c-3p transcript level of PDAC tissues. There was a positive correlation between MALAT-1 and ZEB1 level. MALAT-1 (high)/miR-200c-3p (low) correlated with shorter overall survival of PDAC patients. Multivariate analysis revealed that both MALAT-1 and miR-200c-3p levels were independent prognostic factors.Conclusion: Our findings firstly revealed a novel feedback loop between high MALAT-1 and low miR-200c-3p. Targeting the feedback loop between high MALAT-1 and low miR-200c-3p will be a therapeutic strategy for PDAC. [ABSTRACT FROM AUTHOR]

Background: Emerging studies show that long noncoding RNAs (lncRNAs) play important roles in carcinogenesis and cancer progression. The lncRNA ZEB1 antisense 1 (ZEB1-AS1) derives from the promoter region of ZEB1 and we still know little about its expressions, roles and mechanisms. Methods: RACE was used to obtain the sequence of ZEB1-AS1. RNA interference was used to decrease ZEB1-AS1 expression. Adenovirus expression vector was used to increase ZEB1-AS1 expression. CHIP and RIP were used to detect the epigenetic mechanisms by which ZEB1-AS1 regulated ZEB1. CCK8 assay, wound healing assay and transwell assay were used to measure proliferation and migration of prostate cancer cells. Results: In this study, in prostate cancer cells, we found that RNAi-mediated downregulation of ZEB1-AS1 induced significant ZEB1 inhibition while artificial overexpression of ZEB1-AS1 rescued ZEB1 expression, which means that ZEB1-AS1 promotes ZEB1 expression. Also, ZEB1-AS1 indirectly inhibited miR200c, the well-known target of ZEB1, and upregulated miR200c's target BMI1. Mechanistically, ZEB1-AS1 bound and recruited histone methyltransferase MLL1 to the promoter region of ZEB1, induced H3K4me3 modification therein, and activated ZEB1 transcription. Biologically, ZEB1-AS1 promoted proliferation and migration of prostate cancer cells. Conclusions: Collectively, ZEB1-AS1 functions as an oncogene in prostate cancer via epigenetically activating ZEB1 and indirectly regulating downstream molecules of ZEB1. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence has indicated an important role for estrogen receptor beta 1 (ERβ1) in breast cancer. However, the role of ERβ1 in the metastasis of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) and the underlying mechanisms are still unknown. Methods: Stable ERβ1-expressing TNBC cell lines were generated for this study. We detected the abilities of cell migration and invasion by wound-healing and transwell assays and the expression of E-cadherin and N-cadherin by quantitative RT-PCR (qRT-PCR) and western blotting assays in TNBC cell lines. Chromatin immunoprecipitation (ChIP) analysis was performed to assess the effect of AR on ERβ1 promoter. Tumor metastasis was evaluated in vivo using a lung metastasis mouse model. Lastly, immunohistochemical expression of ERβ1 in TNBC tissues was analyzed and correlated with clinicopathological features. Results: ERβ1 suppressed the invasion and migration abilities of AR-positive TNBC cells and induced the downregulation of ZEB1. ZEB1 overexpression abrogated the increase in E-cadherin expression and the decrease in N-cadherin expression modulated by ERβ1. A lung metastasis mouse model showed that the incidence of metastasis was lower in ERβ1-expressing TNBC cells. Further, AR activation increased the anti-metastatic effect of ERβ1 in AR-positive TNBC cells, which accelerated ERβ1 transcription by functioning as a transcription factor that bound to the promoter of ERβ1. No significant change was observed in AR expression induced by ERβ1. Immunohistochemistry (IHC) analysis of TNBC clinical samples showed that ERβ1 and AR were positive in 31. 7% and 23.2% of samples, respectively. ERβ1 expression was negatively correlated with ZEB1 expression and lymph node metastasis, and positively correlated with the expression of AR and E-cadherin. Conclusion: Our findings suggested a potential role of ERβ1 in metastasis of AR-positive TNBC and provided novel insights into the mechanism of action of ERβ1 and the possible relationship between ERβ1 and AR. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) play important roles in cancer initiation and development. Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and metastasis. The purpose of the study was to determine the function and mechanism of miR-484 in initiation and development of cervical cancer (CC). Methods: We determined the expression levels of miR-484 in cervical cancer tissues and cell lines with RT-qPCR. Prediction algorithms and EGFP reporter assay were performed to evaluate the targets for miR-484. MTT assay, colony formation assay, flow cytometric analysis, transwell cell migration and invasion assays, and detection of EMT markers were employed to investigate the roles of miR-484 and the targets in regulation of cell proliferation and EMT process. We also used rescue experiments to confirm the effect of miR-484 on CC cells through directly regulating the expression of its targets. Results: Firstly we found miR-484 was down-regulated in cervical cancer tissues and cell lines compared with their matched non-cancerous tissues or normal cervical keratinocytes cells. Further studies revealed that overexpression of miR-484 suppressed the cell proliferation, while exacerbates apoptosis. Besides, miR-484 suppressed cellular migration, invasion and EMT process of CC cells. EGFP reporter assay showed that miR-484 binds to ZEB1 and SMAD2 3'UTR region and reduced their expression. The expression of miR-484 had reverse correlation with SMAD2/ZEB1, and SMAD2/ZEB1 had positive correlation with each other in cervical cancer tissues and cell lines. Furthermore, the ectopic expression of ZEB1 or SMAD2 could rescue the malignancies suppressed by miR-484, suggesting that miR- 484 down-regulates ZEB1 and SMAD2 to repress tumorigenic activities. Conclusion: We found miR-484 inhibits cell proliferation and the EMT process by targeting both ZEB1 and SMAD2 genes and functions as a tumor suppressor, which may served as potential biomarkers for cervical cancer. [ABSTRACT FROM AUTHOR]

Background: The aberrant expression of miR-23b is involved in the development and progression of cancer. The aim of this study was to evaluate the potential role of methylation in the silencing of miR-23b in cervical cancer cell lines and to determine its expression in stages of malignant progression and in cervical cancer tissues HPV16-positive. Methods: The methylation of the miR-23b promoter was determined in HeLa, SiHa, CaSki and C33A cells using a Human Cancer miRNA EpiTectMethyl II Signature PCR Array®. The cells were treated with 5-Aza-2′-deoxycytidine, and the expression of miR-23b, uPa, c-Met and Zeb1 was determined by qRT-PCR. miR-92a and GAPDH were used as controls. The expression of miR-23b was determined in cervical scrapes and biopsies of women without squamous intraepithelial lesions, with precursor lesions and with cervical cancer, all were HPV16-positive. The Fisher exact and Mann-Whitney tests were used to compare the differences of the expression of miR-23b, uPa, c-Met and Zeb1 among cell groups, and the difference among patients, respectively. The association between the expression of miR-23b and cervical cancer was determined by logistic regression with a confidence level of 95 %. A value of p < 0.05 was considered statistically significant. Results: In C33A, HeLa and CaSki cells, methylation was associated with decreased expression of miR-23b. After treatment with 5-Aza-CdR, the expression of miR-23b increased in all cell lines and the expression of c-Met decreased in HeLa cells, while uPa and Zeb1 decreased in C33A and CaSki cells. In SiHa cells the expression of uPa, c-Met and Zeb1 increased. The expression of miR-23b decreased in relation to the increase in the severity of the lesion and was significantly lower in cervical cancer. In women with premalignant lesions HPV16-positive, decreased levels of miR-23b increased the risk of cervical cancer (OR = 36, 95 % CI = 6.7-192.6, p < 0.05). Conclusions: The results suggest that the expression of miR-23b is regulated by the methylation of its promoter and is possible that this microRNA influence the expression of uPa, c-Met and Zeb1 in cervical cancer cells lines. In women with premalignant lesions and cervical cancer infected with HPV16, the expression level of miR-23b agree with a tumor suppressor gene. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a common malignant tumor that severely threatens human health. The poor prognosis of HCC is mainly attributed to intrahepatic and extrahepatic metastases. HOXD9 proteins belong to a superfamily that regulates the development and control of many cellular processes, including proliferation, apoptosis, cell shape, and cell migration. HOXD9 can also function as an oncogene in several cancer cells. However, its biological function in human HCC requires further investigation. In this study, HOXD9 exhibited high expression in invasive HCC cells. HOXD9 overexpression can significantly enhance HCC cell migration, invasion, and metastasis, whereas silencing HOXD9 inhibits these processes. HOXD9 also promotes the epithelial--mesenchymal transition (EMT) of HCC cells. Microarray analysis suggests that ZEB1 can function as a downstream factor of HOXD9. HOXD9 can interact with the promoter region of ZEB1 and promotes ZEB1 expression. ZEB1 knockdown inhibits HOXD9- induced migration and invasion, as well as EMT in HCC cells. This study helps elucidates the oncogenic functions of HOXD9 in HCC. [ABSTRACT FROM AUTHOR]

Background: Previous studies identified microRNAs (miRNAs) and messenger RNAs with significantly different expression between normal pancreas and pancreatic cancer (PDAC) tissues. Due to technological limitations of microarrays and real-time PCR systems these studies focused on a fixed set of targets. Expression of other RNA classes such as long intergenic non-coding RNAs or sno-derived RNAs has rarely been examined in pancreatic cancer. Here, we analysed the coding and non-coding transcriptome of six PDAC and five control tissues using next-generation sequencing. Results: Besides the confirmation of several deregulated mRNAs and miRNAs, miRNAs without previous implication in PDAC were detected: miR-802, miR-2114 or miR-561. SnoRNA-derived RNAs (e.g. sno-HBII-296B) and piR-017061, a piwi-interacting RNA, were found to be differentially expressed between PDAC and control tissues. In silico target analysis of miR-802 revealed potential binding sites in the 3' UTR of TCF4, encoding a transcription factor that controls Wnt signalling genes. Overexpression of miR-802 in MiaPaCa pancreatic cancer cells reduced TCF4 protein levels. Using Massive Analysis of cDNA Ends (MACE) we identified differential expression of 43 lincRNAs, long intergenic non-coding RNAs, e.g. LINC00261 and LINC00152 as well as several natural antisense transcripts like HNF1A-AS1 and AFAP1-AS1. Differential expression was confirmed by qPCR on the mRNA/miRNA/lincRNA level and by immunohistochemistry on the protein level. Conclusions: Here, we report a novel lncRNA, sncRNA and mRNA signature of PDAC. In silico prediction of ncRNA targets allowed for assigning potential functions to differentially regulated RNAs. [ABSTRACT FROM AUTHOR]

Background: Chemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear. Methods: Bioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed. Results: ZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients. Conclusion: Our finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1–CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease. [ABSTRACT FROM AUTHOR]

Background: Ras pathway mutation leads to induction and Erk phosphorylation and activation of the Ets1 transcription factor. Ets1 in turn induces cyclin E and cyclin dependent kinase (cdk) 2 to drive cell cycle progression. Ets1 also induces expression of the epithelial-mesenchymal transition (EMT) transcription factor Zeb1, and thereby links Ras mutation to EMT, which is thought to drive tumor invasion. Ras pathway mutations are detected by the Rb1 tumor suppression pathway, and mutation or inactivation of the Rb1 pathway is required for EMT. Results: We examined linkage between Rb1, Ets1 and Zeb1. We found that an Rb1-E2F complex binds the Ets1 promoter and constitutively limits Ets1 expression. But, Rb1 repression of Zeb1 provides the major impact of Rb1 on Ets1 expression. We link Rb1 repression of Zeb1 to induction of miR-200 family members, which in turn target Ets1 mRNA. These findings suggest that Ets1 and Zeb1 comprise an amplification loop that is dependent upon miR-200 and regulated by Rb1. Thus, induction of Ets1 when the Rb1 pathway is lost may contribute to deregulated cell cycle progression through Ets1 induction of cyclin E and cdk2. Consistent with such an amplification loop, we correlate expression of Ets1 and Zeb1 in mouse and human lung adenocarcinoma. In addition we demonstrate that Ets1 expression in thymocytes is also dependent upon Zeb1. Conclusions: Taken together, our results provide evidence of an Rb1-dependent Ets1-Zeb1 amplification loop in thymocyte differentiation and tumor invasion. [ABSTRACT FROM AUTHOR]

Background Transforming growth factor beta (TGF-β) plays major roles in tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis. The epithelial markers E-cadherin, claudin-3 and claudin-4, commonly decreased in human adenocarcinomas are actually up regulated during ovarian carcinogenesis. In human ovarian cancer TGF-β1 may either suppress or promote tumor progression, but whether other TGF-β isoforms (TGF-β2 and TGF-β3) exert similar effects is not known. Methods In this study we investigated the ability of the TGF-β isoforms (TGF-β1-3) to induce proliferation and migration by BrdU labeling, scratch wound and trans-filter migration assays in the human serous adenocarcinoma cell-line NIH-OVCAR3. Transepithelial resistance was measured and EMT observed by light-microscopy. Expression of adherens-, tight-junction and EMT-related transcription factors was analyzed by qRT-PCR and immunoblotting. Results All TGF-β isoforms dose-dependently inhibited NIH-OVCAR3 cell growth, stimulated tumor cell migration with similar efficiency. The mesenchymal marker N-cadherin and claudin-1 expression was induced and occludin down regulated. However, migrating cells retained an epithelial shape and E-cadherin expression. The E-cadherin repressor SNAIL mRNA levels remained low independently of TGF-β1-3 treatment while ZEB1 expression was enhanced. Conclusions TGF-β1, TGF-β2 and TGF-β3 promote migration of NIH-OVCAR3 ovarian cancer cells independently of cell proliferation and without conversion to a complete EMT phenotype. Epithelial ovarian cancer commonly metastasis to the surrounding tissue or inside the peritoneum rather than invading blood vessels to set distance metastasis. Our result raises the question whether ovarian cancer primarily spread via collective migration than via single cell invasion. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown causes. Three proteins (mammalian target of rapamycin, mTOR; zinc finger E-box-binding homeobox 1, ZEB1; Rho-associated, coiled-coil containing protein kinase 1, ROCK1) may be related to pulmonary fibrosis. However, they have not been assessed in human pulmonary fibrosis. We assessed the clinical significance of mTOR, ZEB1, and ROCK1 expression in human pulmonary fibrosis of usual interstitial pneumonia (UIP) pattern. Methods: The mTOR, ZEB1, and ROCK1 expression was evaluated by immunohistochemical staining of 30 surgical lung biopsy tissues from 26 IPF and 4 UIP pattern connective tissue disease related interstitial lung disease (CTD-ILD) patients. The expression scores correlated with the clinical features. Results: The mTOR, ZEB1 and ROCK1 mainly expressed in alveolar epithelial cells of UIP lungs. The histological fibrosis scores and lung function decline in the strong mTOR expression group were higher than those in the weak and intermediate expression group. Patients with positive ZEB1 expression had higher fibrosis scores and greater decline in carbon monoxide diffusion capacity (DLCO) than patients with negative ZEB1 expression. Patients with positive mTOR or ZEB1 expression had poorer prognosis than that of patients with negative mTOR or ZEB1 expression, although it was not statistically significant. ROCK1 was not associated with the studied clinicopathological features. Conclusions: The mTOR and ZEB1 expression in pulmonary fibrosis patients significantly correlated with the fibrosis score and lung function decline, indicating that it may be related to the prognosis of pulmonary fibrosis. Further studies involving large numbers of homogeneous IPF patients are warranted. [ABSTRACT FROM AUTHOR]

Background: IL-1β is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1β may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1β in colon cancer stem cell (CSC) development. Methods: Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1β on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116. Results: We found that IL-1β can increase sphere-forming capability of colon cancer cells in serum free medium. IL-1β-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1β-induced spheres, indicating that there might be a close association between EMT and IL-1β-induced CSC self-renewal. Indeed, IL-1β treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1β-induced EMT and stem cell formation. Conclusion: Our findings indicate that IL-1β may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1β and Zeb1 might be new therapeutic targets against colon cancer stem cells. [ABSTRACT FROM AUTHOR]