Zinc Finger E-box-binding Homeobox 2 Overexpression Research Articles

MiR-141-3p protects against blood–brain barrier disruption and brain injury after intracerebral hemorrhage by targeting ZEB2

MicroRNAs (miRNAs) participate in the diagnosis and treatment of intracerebral hemorrhage (ICH). miR-141-3p has been widely reported to regulate neurological disorders and cerebropathy. However, the specific role of miR-141-3p in ICH has not yet been revealed. The aim of this study was exploration of the biological functions and mechanism of miR-141-3p in ICH by establishing a collagenase-induced ICH mouse model. After ICH induction, miR-141-3p mimics or miR-NC were administered into the right striatum of the model mice followed by the performance of neurological tests. After euthanasia of the mice, the injury volume, brain water content, and injury to the blood–brain barrier (BBB) were evaluated. Evans blue (EB) was used to stain the brain slices, and EB extravasation was detected to evaluate the injury to BBB. miR-141-3p expression in perihematomal edema and hematoma areas after ICH was assessed by RT–qPCR. The levels of tight junction proteins in brain tissues and human brain microvascular endothelial cells (BMECs) were evaluated by western blotting. The FITC-dextran 20 method was used to assess BMEC permeability. The binding between miR-141-3p and zinc finger E-box-binding homeobox 2 (ZEB2) was verified with a luciferase reporter assay. In this study, miR-141-3p overexpression alleviated ICH-induced brain injury and protected BBB integrity in vivo. ZEB2 was a target gene of miR-141-3p. ZEB2 overexpression promoted BBB disruption, and miR-141-3p overexpression attenuated the promoting effect exerted by ZEB2. Overall, miR-141-3p protects against BBB disruption and attenuates brain injuries induced by ICH by targeting ZEB2.

Puerarin Prevents Acute Liver Injury via Inhibiting Inflammatory Responses and ZEB2 Expression.

Puerarin, an isoflavone component extracted from herb radix puerariae, is widely used in China in the treatment of immune diseases and inflammation. Previous studies have demonstrated that puerarin prevented acute lung injury by regulating inflammatory responses. However, the effect of puerarin on acute liver injury (ALI) was unclear. The purpose of this study was to explore the beneficial effects of puerarin when applied to ALI. We found that puerarin inhibited liver injury and inflammatory cell infiltration in lipopolysaccharide (LPS)/D-galactose (D-Gal)-induced acute liver failure and the liver pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in liver tissues with ALI and LPS-induced L-02 cells but upregulated the expression level of zinc finger E-box-binding homeobox 2 (ZEB2). Significantly, the results of this study showed that the inhibition of liver pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) production in LPS-induced L-02 cells was caused by ZEB2 overexpression. However, knocking down ZEB2 promoted LPS-mediated secretion of liver pro-inflammatory cytokines in L-02 cells. Additional experiments showed that puerarin inhibited the activation of the NF-κB signaling pathway by elevating ZEB2 expression in L-02 cells. In summary, puerarin most likely prevented activation of the pro-inflammatory factors and reduced LPS/D-Gal-induced liver injury by enhancing the ZEB2 expression level and, consequently, blocking activation of the NF-κB signaling pathway in the liver.

Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies.

ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

MicroRNA-140 Represses Esophageal Cancer Progression via Targeting ZEB2 to Regulate Wnt/β-Catenin Pathway

BackgroundMicroRNAs have been reported to play regulatory functions in various cancers, including esophageal cancer. The aim of this study was to investigate the effects of miR-140 on the progression of esophageal cancer and the underlying regulatory mechanism. MethodsThe levels of miR-140 and zinc finger E-box-binding homeobox 2 (ZEB2) messenger RNA in esophageal cancer tissues and cell lines were measured by quantitative real-time polymerase chain reaction. The protein levels of ZEB2, β-catenin, c-Myc, and cyclinD1 were determined by Western blot. Cell proliferation and apoptosis were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and flow cytometry, respectively. Cell migration and invasion were assessed by transwell assay. In addition, the relationship between miR-140 and ZEB2 was predicted by TargetScan online database and confirmed by dual-luciferase reporter assay. The tumor xenograft model was used to verify the role of miR-140 in esophageal cancer progression in vivo. ResultsThe expression of miR-140 was downregulated whereas ZEB2 expression was upregulated in esophageal cancer tissues compared with paracancerous normal tissues. Functionally, both miR-140 overexpression and ZEB2 knockdown inhibited proliferation, migration, and invasion and induced apoptosis in esophageal cancer cells. ZEB2 overexpression reversed the effects of miR-140 on proliferation, apoptosis, migration, and invasion of esophageal cancer cells. Mechanistically, ZEB2 was identified as a target of miR-140. Furthermore, miR-140 suppressed Wnt/β-catenin pathway by regulating ZEB2 expression in esophageal cancer cells. MiR-140 inhibited tumor growth of esophageal cancer through repressing ZEB2 expression in vivo. ConclusionsOur results demonstrated that miR-140 inhibited esophageal cancer development by targeting ZEB2 through inactivating Wnt/β-catenin pathway.

Hypoxia‐inhibited miR‐338‐3p suppresses breast cancer progression by directly targeting ZEB2

Hypoxia plays an essential role in the development of various cancers. The biological function and underlying mechanism of microRNA‐338‐3p (miR‐338‐3p) under hypoxia remain unclarified in breast cancer (BC). Herein, we performed bioinformatics, gain and loss of function of miR‐338‐3p, a luciferase reporter assay, and chromatin immunoprecipitation (ChIP) in vitro and in a tumor xenograft model. We also explored the potential signaling pathways of miR‐338‐3p in BC. We detected the expression levels and prognostic significance of miR‐338‐3p in BC by qRT‐PCR and in situ hybridization. MiR‐338‐3p was lowly expressed in BC tissues and cell lines, and BC patients with underexpression of miR‐338‐3p tend to have a dismal overall survival. Functional experiments showed that miR‐338‐3p overexpression inhibited BC cell proliferation, invasion, migration, and epithelial‐mesenchymal transition (EMT) process, whereas miR‐338‐3p silencing abolished these biological behaviors. Zinc finger E‐box‐binding homeobox 2 (ZEB2) was validated as a direct target of miR‐338‐3p. ZEB2 overexpression promoted while ZEB2 knockdown abolished the promoted effects of miR‐338‐3p knockdown on cell biological behaviors through the NF‐ĸB and PI3K/Akt signal pathways. HIF1A can transcriptionally downregulate miR‐338‐3p under hypoxia. In total, miR‐338‐3p counteracts hypoxia‐induced BC cells growth, migration, invasion, and EMT via the ZEB2 and NF‐ĸB/PI3K signal pathways, implicating miR‐338‐3p may be a promising target to treat patients with BC.

CircPRKCI relieves lipopolysaccharide-induced HK2 cell injury by upregulating the expression of miR-545 target gene ZEB2.

The aim of this study was to investigate the possible influences of circPRKCI abnormal expression on lipopolysaccharide (LPS)-induced HK2 cell injury and its mechanism. The circPRKCI level was identified in serum samples from patients with urosepsis and healthy subjects, as well as LPS-treated HK2 cells by qRT-PCR. Cell viability, apoptosis, expression of proteins associated with apoptosis, and expression of pro-inflammatory cytokines in LPS-treated HK2 cells were measured. Effects of circPRKCI abnormal expression on LPS-induced HK2 cell injury were then evaluated. Afterward, the binding miRNA of circPRKCI and target gene of miRNA were identified, and the involvements of NF-kB pathway signaling pathway with the effects of circPRKCI were finally studied. CircPRKCI was significantly down-regulated in serum samples from patients with urosepsis and LPS-treated HK2 cells. LPS-induced decrease of cell viability, increase of cell apoptosis, as well as elevated productions of tumor necrosis factor (TNF)-α, interleukins (IL)-1β, IL-6, and IL-8 in HK2 cells were attenuated by overexpressed circPRKCI. In addition, circPRKCI negatively regulated the expression of miR-545, and miR-545 up-regulation reversed the inhibiting effects of circPRKCI overexpression on LPS-induced HK2 cell injury. Moreover, zinc finger E-box-binding homeobox 2 (ZEB2) was identified as a target gene of miR-545, and ZEB2 overexpression partly reversed the effects of miR-545 up-regulation on LPS-induced HK2 cell injury. Furthermore, NF-kB pathway was revealed to be associated to the effects of circPRKCI on LPS-induced HK2 cell injury. This research indicated that the highly expressed circPRKCI relieved inflammatory injury induced by LPS in HK2 cells by suppressing miR-545/ZEBs and depressing the briskness of NF-kB pathway.

MicroRNA-377 Counteracts With Cancer Stem Cell Phenotypes and Epithelial Mesenchymal Transformation by Targeting ZEB2 in Colon Cancer.

Dysregulated microRNAs (miRNAs) have been implicated in the pathogenic processes of colon cancer. Epithelial mesenchymal transition (EMT) promotes metastatic progression and cancer stem cells are closely involved in colon cancer proliferation and metastasis. Functional effects of miR-377 on colon cancer stem cell phenotypes and EMT were then determined in the present study. Firstly, reduced miR-377 was found in colon cancer tissues and cell lines. Results from flow cytometry, sphere formation and western blot assays showed that miR-377 knockdown increased number of ALDH+ cells and promoted sphere formation ability. Moreover, cell migration/invasion and EMT of colon cancer cells were suppressed by miR-377 over-expression. On the contrary, miR-377 mimics caused the reversed results. ZEB2 (zinc finger E box-binding homeobox 2) was then validated as a binding target of miR-377. ZEB2 over-expression reversed the inhibitory abilities of miR-377 on cancer stem cell phenotypes, EMT, migration and invasion. In conclusion, miR-377 regulates cancer stem cell phenotypes and EMT in colon cancer cells via regulation of ZEB2, suggesting a new therapeutic strategy for colon cancer treatment.

Upregulation Of miR-153 Inhibits Triple-Negative Breast Cancer Progression By Targeting ZEB2-Mediated EMT And Contributes To Better Prognosis.

BackgroundTriple-negative breast cancer (TNBC) is the most malignant type of breast cancer. MicroRNAs (miRs) and their corresponding molecular targets are associated with the occurrence and development of various human malignancies. However, the roles of the microRNA-153 (miR-153) and zinc finger E-box-binding homeobox 2 (ZEB2)-induced epithelial–mesenchymal transition (EMT) in TNBC and predictive effect of miR-153 on the prognosis of TNBC have not been fully elucidated.Materials and methodsRelative miR-153 expression level was examined by RT-qPCR assay in TNBC tissues of 60 patients and TNBC cell lines (SKBR3, BT-549 and MDA-MB-231). Cell proliferation ability, invasion ability and migration ability were measured by CCK8 assay, Transwell invasion assay and wound healing assay, respectively. Luciferase reporting experiment was used to confirm that there was a miR-153-binding site in ZEB2 3ʹ-UTR. The expression of ZEB2 in tissues and its relationship with miR-153 were analyzed with immunohistochemistry method. Relative ZEB2, E-cadherin, N-cadherin and Vimentin mRNA and protein expression levels were observed with RT-qPCR and Western blot, respectively. Based on risk factors, a prognostic model was established according to the Cox proportional risk model, and the prognostic risk factors of TNBC patients were predicted and analyzed.ResultsThe expression of miR-153 in TNBC tissues and cells was declined (all P<0.01), and upregulation of miR-153 inhibited proliferation, invasion and migration of TNBC cells (all P<0.01). In addition, miR-153 regulated ZEB2/EMT link in TNBC, and ZEB2 overexpression reversed the tumor-suppressive effect of miR-153 in TNBC. Moreover, miR-153 was an independent predictive factor that was associated with excellent prognosis in TNBC patients.ConclusionmiR-153 may inhibit TNBC proliferation, invasion and migration by regulating ZEB2/EMT link. Therefore, miR-153 is expected to be a molecular target and prognostic marker for TNBC.