CircAGFG1 Promotes Ovarian Cancer Progression Through the miR-409-3 p/ZEB1 Axis.

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.

Extracellular matrix marker LAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

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.

Ethanol responsive lnc171 promotes migration and invasion of HCC cells via mir-873-5p/ZEB1 axis.

BACKGROUNDS: Long nonconding RNAs (lncRNAs) have been found to be a vital regulatory factor in the development process of human cancer, and could regarded as diagnostic or prognostic biomarkers for human cancers. Here, we aim to confirm the expression and molecular mechanism of RP11-171K16.5 (lnc171) in hepatocellular carcinoma (HCC). METHODS: Screening of differentially expressed lncRNAs by RNA sequencing. Expression level of gene was studied by quantitative real-time PCR (qRT-PCR). The effects of lnc171, mir-873-5p, and ethanol on migration and invasion activity of cells were studied used transwell assay, and luciferase reporter assay was used to confirm the binding site. RESULTS: RNA sequencing showed that lnc171 was markedly up-regulated in HCC. siRNA-mediated knockdown of lnc171 repressed the migration and invasion ability of HCC cells. Bioinformatic analysis, dual luciferase reporter assay, and qRT-PCR indicated that lnc171 interacted with mir-873-5p in HCC cells, and Zin-finger E-box binding homeobox (ZEB1) was a downstream target gene of mir-873-5p. In addition, lnc171 could enhance migration and invasion ability of HCC cells by up-regulating ZEB1 via sponging mir-873-5p. More interestingly, ethanol stimulation could up-regulate the increase of lnc171, thereby regulating the expression of competing endogenous RNA (ceRNA) network factors which lnc171 participated in HCC cells. CONCLUSIONS: Our date demonstrates that lnc171 was a responsive factor of ethanol, and plays a vital role in development of HCC via binding of mir-873-5p.

Effects of Mesenchymal Stem Cells-Derived Extracellular Vesicles on Inhibition of Hepatic Fibrosis by Delivering miR-200a.

BACKGROUND: Hepatic fibrosis (HF) is a common pathological feature of chronic hepatic diseases. We aimed to illuminate the significance of amniotic mesenchymal stem cells (AMSCs)-derived extracellular vesicles (AMSCs-EVs) in HF. METHODS: Human AMSCs-EVs were isolated and identified. HF mice were constructed and treated with EVs. The fibrosis was observed by staining experiments and Western blot (WB) assay. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and hepatic hydroxyproline (Hyp) were detected to confirm liver function. For the in vitro experiments, human hepatic stellate cells were induced with transforming growth factor-ß and treated with EVs. To measure the degree of HF, the expression of alpha-smooth muscle actin (α-SMA) and Collagen I was detected by WB assay, and cell proliferation was detected by cell counting kit 8 assay. The levels of miR-200a, Zinc finger E-box binding homeobox 1 (ZEB1), and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were detected by WB and real-time quantitative polymerase chain reaction. The binding of ZEB1 to PIK3R3 and miR-200a to ZEB1 was analyzed by chromatin immunoprecipitation and dual luciferase assays to validate their relationships. RESULTS: Human AMSCs and AMSCs-EVs were obtained. Serum ALT, AST, TBIL, and hepatic Hyp were increased, implying the fibrosis degree was aggravated in HF mice, which was decreased again after EV treatment. EVs inhibited HF degree by reducing α-SMA and Collagen I and promoting cell proliferation. AMSCs-EVs delivered miR-200a into hepatocytes, which up-regulated miR-200a expression, inhibited ZEB1 expression, and reduced its enrichment on the PIK3R3 promoter, therefore inhibiting PIK3R3 expression and alleviating HF. Overexpression of ZEB1 or PIK3R3 attenuated the anti-fibrotic effect of AMSCs-EVs. CONCLUSION: Human AMSCs-derived EVs mediated miR-200a delivery and inhibition of intracellular ZEB1/PIK3R3 axis to exert anti-fibrosis effects.

EIF4A3 modulated circ_000999 promotes epithelial-mesenchymal transition in cadmium-induced malignant transformation through the miR-205-5p/ZEB1 axis.

Cadmium (Cd) is an accumulative toxic metal which poses a serious threat to human health, even in trace amounts. One of the most important steps in the pathophysiology of lung cancer (LC) is the epithelial-mesenchymal transition (EMT). In this investigation, a cell malignant transformation model was established by exposing human bronchial epithelial cells (16HBE) to a low dose of Cd for 30 weeks, after which a highly expressed circular RNA (circ_000999) was identified. Cd-induced EMT was clearly observed in rat lungs and 16HBE cells, which was further enhanced following circ_000999-overexpression. Furthermore, upregulated EIF4A3 interacted with the parental gene AGTPBP1 to promote high expression of circ_000999. Subsequent experiments confirmed that circ_000999 could regulate the EMT process by competitively binding miR-205-5p and inhibiting its activity, consequently upregulating expression of zinc finger E-box binding protein 1 (ZEB1). Importantly, the circ_000999 expression level in LC tissues was significantly increased, exhibiting a strong correlation with EMT indicators. Overall, these findings provide a new objective and research direction for reversing lung EMT and subsequent treatment and prevention of LC.

ZEB1 controls a lineage-specific transcriptional program essential for melanoma cell state transitions.

Cell plasticity sustains intra-tumor heterogeneity and treatment resistance in melanoma. Deciphering the transcriptional mechanisms governing reversible phenotypic transitions between proliferative/differentiated and invasive/stem-like states is required. Expression of the ZEB1 transcription factor is frequently activated in melanoma, where it fosters adaptive resistance to targeted therapies. Here, we performed a genome-wide characterization of ZEB1 transcriptional targets, by combining ChIP-sequencing and RNA-sequencing, upon phenotype switching in melanoma models. We identified and validated ZEB1 binding peaks in the promoter of key lineage-specific genes crucial for melanoma cell identity. Mechanistically, ZEB1 negatively regulates SOX10-MITF dependent proliferative/melanocytic programs and positively regulates AP-1 driven invasive and stem-like programs. Comparative analyses with breast carcinoma cells revealed lineage-specific ZEB1 binding, leading to the design of a more reliable melanoma-specific ZEB1 regulon. We then developed single-cell spatial multiplexed analyses to characterize melanoma cell states intra-tumoral heterogeneity in human melanoma samples. Combined with scRNA-Seq analyses, our findings confirmed increased ZEB1 expression in Neural-Crest-like cells and mesenchymal cells, underscoring its significance in vivo in both populations. Overall, our results define ZEB1 as a major transcriptional regulator of cell states transitions and provide a better understanding of lineage-specific transcriptional programs sustaining intra-tumor heterogeneity in melanoma.

GINS1 promotes ZEB1-mediated epithelial-mesenchymal transition and tumor metastasis via ß-catenin signaling in hepatocellular carcinoma.

GINS1 regulates DNA replication in the initiation and elongation phases and plays an important role in the progression of various malignant tumors. However, the role of GINS1 in hepatocellular carcinoma (HCC) remains largely unclear. In this study, we investigated the role and underlying mechanisms of GINS1 in contributing to HCC metastasis. We found that GINS1 was significantly upregulated in HCC tissues and cell lines, especially in HCC tissues with vascular invasion and HCC cell lines with highly metastatic properties. Additionally, high expression of GINS1 was positively correlated with the progressive clinical features of HCC patients, including tumor number (multiple), tumor size (>5 cm), advanced tumor stage, vascular invasion and early recurrence, suggesting that GINS1 upregulation was greatly involved in HCC metastasis. Moreover, Kaplan-Meier survival analysis revealed that high GINS1 expression predicted a poor prognosis. Both in vitro and in vivo, silencing of GINS1 inhibited proliferation, migration, invasion and metastasis, while overexpression of GINS1 induced opposite effects. Mechanistically, we found that ZEB1 was a crucial regulator of GINS1-induced epithelial-mesenchymal transition (EMT), and GINS1 promoted EMT and tumor metastasis through ß-catenin signaling. Overall, the present study demonstrated that GINS1 promoted ZEB1-mediated EMT and tumor metastasis via ß-catenin signaling in HCC.

The deubiquitinase BRCC3 increases the stability of ZEB1 and promotes the proliferation and metastasis of triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) has a high recurrence rate, metastasis rate and mortality rate. The aim of this study is to identify new targets for the treatment of TNBC. Clinical samples are used for screening deubiquitinating enzymes (DUBs). MDA-MB-231 cells and a TNBC mouse model are used for in vitro and in vivo experiments, respectively. Western blot analysis is used to detect the protein expressions of DUBs, zinc finger E-box binding homeobox 1 (ZEB1), and epithelial-mesenchymal transition (EMT)-related markers. Colony formation and transwell assays are used to detect the proliferation, migration and invasion of TNBC cells. Wound healing assay is used to detect the mobility of TNBC cells. Immunoprecipitation assay is used to detect the interaction between breast cancer susceptibility gene 1/2-containing complex subunit 3 (BRCC3) and ZEB1. ZEB1 ubiquitination levels, protein stability, and protein degradation are also examined. Pathological changes in the lung tissues are detected via HE staining. Our results show a significant positive correlation between the expressions of BRCC3 and ZEB1 in clinical TNBC tissues. Interference with BRCC3 inhibits TNBC cell proliferation, migration, invasion and EMT. BRCC3 interacts with ZEB1 and interferes with BRCC3 to inhibit ZEB1 expression by increasing ZEB1 ubiquitination. Interference with BRCC3 inhibits TNBC cell tumorigenesis and lung metastasis in vivo. In all, this study demonstrates that BRCC3 can increase the stability of ZEB1, upregulate ZEB1 expression, and promote the proliferation, migration, invasion, EMT, and metastasis of TNBC cells, providing a new direction for cancer therapy.

YAP/TAZ-mediated regulation of laminin 332 is enabled by ß4 integrin repression of ZEB1 to promote ferroptosis resistance.

We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.

Combinatorial Cooperativity in miR200-Zeb Feedback Network can Control Epithelial-Mesenchymal Transition.

Carcinomas often utilize epithelial-mesenchymal transition (EMT) programs for cancer progression and metastasis. Numerous studies report SNAIL-induced miR200/Zeb feedback circuit as crucial in regulating EMT by placing cancer cells in at least three phenotypic states, viz. epithelial (E), hybrid (h-E/M), mesenchymal (M), along the E-M phenotypic spectrum. However, a coherent molecular-level understanding of how such a tiny circuit controls carcinoma cell entrance into and residence in various states is lacking. Here, we use molecular binding data and mathematical modeling to report that the miR200/Zeb circuit can essentially utilize combinatorial cooperativity to control E-M phenotypic plasticity. We identify minimal combinatorial cooperativities that give rise to E, h-E/M, and M phenotypes. We show that disrupting a specific number of miR200 binding sites on Zeb as well as Zeb binding sites on miR200 can have phenotypic consequences-the circuit can dynamically switch between two (E, M) and three (E, h-E/M, M) phenotypes. Further, we report that in both SNAIL-induced and SNAIL knock-out miR200/Zeb circuits, cooperative transcriptional feedback on Zeb as well as Zeb translation inhibition due to miR200 are essential for the occurrence of intermediate h-E/M phenotype. Finally, we demonstrate that SNAIL can be dispensable for EMT, and in the absence of SNAIL, the transcriptional feedback can control cell state transition from E to h-E/M, to M state. Our results thus highlight molecular-level regulation of EMT in miR200/Zeb circuit and we expect these findings to be crucial to future efforts aiming to prevent EMT-facilitated dissemination of carcinomas.

The mechanism of PFK-1 in the occurrence and development of bladder cancer by regulating ZEB1 lactylation.

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.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer.

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3'-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Long Non-coding RNA X-Inactive Specific Transcript Promotes Esophageal Squamous Cell Carcinoma Progression via the MicroRNA 34a/Zinc Finger E-box-Binding Homeobox 1 Pathway.

BACKGROUND: The long non-coding RNA X-inactive specific transcript (XIST) plays a crucial role in transcriptional silencing of the X chromosome. Zinc finger E-box-binding homeobox 1 (ZEB1) is a transcription factor involved in epithelial-mesenchymal transition (EMT) regulation. AIMS: This study aimed to investigate the impact of XIST on esophageal squamous cell carcinoma (ESCC) progression and its underlying mechanism involving the miR-34a/ZEB1/E-cadherin/EMT pathway. METHODS: XIST and ZEB1 expression were analyzed using quantitative PCR and immunohistochemistry. XIST knockdown was achieved in KYSE150 ESCC cells using siRNA or shRNA lentivirus transfection. Proliferation, migration, and invasion abilities were assessed, and luciferase reporter assays were performed to confirm XIST-miR-34a-ZEB1 interactions. In vivo ESCC growth was evaluated using a xenograft mouse model. RESULTS: XIST and ZEB1 were upregulated in tumor tissues, correlating with metastasis and reduced survival. XIST knockdown inhibited proliferation, migration, and invasion of KYSE150 cells. It decreased ZEB1 expression, increased E-cadherin and miR-34a levels. Luciferase reporter assays confirmed miR-34a binding to XIST and ZEB1. XIST knockdown suppressed xenograft tumor growth. CONCLUSION: XIST promotes ESCC progression via the miR-34a/ZEB1/E-cadherin/EMT pathway. Targeting the XIST/miR-34a/ZEB1 axis holds therapeutic potential and serves as a prognostic biomarker in ESCC.

Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis.

BACKGROUND: The dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear. METHODS: Exosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed. RESULTS: Our previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression. CONCLUSIONS: Our results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

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

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease involving multiple factors and genes. Astragaloside IV (ASV) is one of the main bioactive ingredients extracted from the root of Astragalus membranaceus, which plays an important role in anti-inflammatory, antioxidant and improve cardiopulmonary function. Epithelial-mesenchymal transition (EMT) is a key driver of the process of pulmonary fibrosis, and Zinc finger E-box-binding homeobox 1 (ZEB1) can promote pulmonary fibrosis in an EMT-dependent manner. Here, we found that ASV effectively inhibited the ZEB1 and EMT in both bleomycin (BLM)-induced rat pulmonary fibrosis and TGF-ß1-treated A549 cells. To further elucidate the molecular mechanisms underlying effects of ASV in IPF, we explored the truth using bioinformatics, plasmid construction, immunofluorescence staining, western blotting and other experiments. Dual luciferase reporter assay and bioinformatics proved that miR-200c not only acts as an upstream regulatory miRNA of ZEB1 but also has binding sites for the lncRNA-ATB. In A549 cell-based EMT models, ASV reduced the expression of lncRNA-ATB and upregulated miR-200c. Furthermore, overexpression of lncRNA-ATB and silencing of miR-200c reversed the down-regulation of ZEB1 and the inhibition of EMT processes by ASV. In addition, the intervention of ASV prevented lncRNA-ATB as a ceRNA from regulating the expression of ZEB1 through sponging miR-200c. Taken together, the results showed that ASV inhibited the EMT process through the lncRNA-ATB/miR-200c/ZEB1 signaling pathway, which provides a novel approach to the treatment of IPF.

Toosendanin Restrains Idiopathic Pulmonary Fibrosis by Inhibiting ZEB1/CTBP1 Interaction.

BACKGROUND: Extensive deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) is due to hyperactivation and proliferation of pulmonary fibroblasts. However, the exact mechanism is not clear. OBJECTIVE: This study focused on the role of CTBP1 in lung fibroblast function, elaborated its regulation mechanism, and analyzed the relationship between CTBP1 and ZEB1. Meanwhile, the antipulmonary fibrosis effect and its molecular mechanism of Toosendanin were studied. METHODS: Human IPF fibroblast cell lines (LL-97A and LL-29) and normal fibroblast cell lines (LL-24) were cultured in vitro. The cells were stimulated with FCS, PDGF-BB, IGF-1, and TGF-ß1, respectively. BrdU detected cell proliferation. The mRNA expression of CTBP1 and ZEB1 was detected by QRT-PCR. Western blotting was used to detect the expression of COL1A1, COL3A1, LN, FN, and α-SMA proteins. An animal model of pulmonary fibrosis was established to analyze the effects of CTBP1 silencing on pulmonary fibrosis and lung function in mice. RESULTS: CTBP1 was up-regulated in IPF lung fibroblasts. Silencing CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts. Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts. Silencing CTBP1 reduced the degree of pulmonary fibrosis in mice with pulmonary fibrosis. Western blot, CO-IP, and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts. Toosendanin can inhibit the ZEB1/CTBP1protein interaction and further inhibit the progression of pulmonary fibrosis. CONCLUSION: CTBP1 can promote the activation and proliferation of lung fibroblasts through ZEB1. CTBP1 promotes lung fibroblast activation through ZEB1, thereby increasing excessive deposition of ECM and aggravating IPF. Toosendanin may be a potential treatment for pulmonary fibrosis. The results of this study provide a new basis for clarifying the molecular mechanism of pulmonary fibrosis and developing new therapeutic targets.

miR-186 regulates epithelial-mesenchymal transformation to promote nasopharyngeal carcinoma metastasis by targeting ZEB1.

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

Mechanisms of LPS-induced epithelial mesenchymal transition in bEECs.

High-concentrate diets cause subacute ruminal acidosis, resulting in increased blood lipopolysaccharide (LPS) levels in cows. We found that the peak LPS in cows fed with high-concentrate diets coincides the period of embryo implantation in a large-scale dairy farm. As epithelial-mesenchymal transition (EMT) should be tightly regulated during normal embryo implantation in cows, we speculated that increased LPS may cause abnormal EMT, thereby inhibiting embryo implantation in cows. To confirm that elevated LPS levels induce abnormal EMT in cows, we treated bovine endometrial epithelial cells (bEECs) with LPS for 48 h and analyzed the protein levels of ZEB1, a major EMT-related transcription factor, which is positively regulated by the TGFß/SMAD3 pathway. In addition, we analyzed the changes in expression of three EMT-related genes (E-cadherin, N-cadherin, and Vimentin), and examined the morphology and migratory ability of the cells. The results showed that elevated LPS levels increased protein expression of ZEB1, vimentin, and N-cadherin, and reduced that of E-cadherin. Elevated LPS also increased bEECs migration rate, and induced the cells to acquire a mesenchymal phenotype. Furthermore, benzyl butyl phthalate (BBP)-induced ZEB1 overexpression significantly decreased E-cadherin levels and increased N-cadherin levels. As LPS treatment also decreased the expression of Bta-miR-200b, we further found that Bta-miR-200b targets to the 3'UTR of ZEB1 through the confirmation of dual-luciferase reporter system. And the increased level of Bta-miR-200b by mimic enhanced the expression of E-cadherin and yet inhibited the expression of N-cadherin in protein, which exactly opposite to the results induced by LPS. In conclusion, LPS induced EMT in bEECs by upregulating ZEB1, while Bta-miR-200b could inhibit the occurrence of EMT by binding ZEB1 3'UTR. These results provide a new insight for low reproductive rate of dairy cows under the background of high-concentrate diets.

Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1WT/ApoeKO), male mice with Zeb1 ablation in their myeloid cells (Zeb1∆M/ApoeKO) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1∆M/ApoeKO mice display more pronounced systemic metabolic alterations than Zeb1WT/ApoeKO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1∆M/ApoeKO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.