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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]

Simple Summary: Given the importance of the epithelial-to-mesenchymal transition in tumor progression and the pivotal role of ZEB1 as a transcriptional repressor during this process, the regulation of ZEB1-targeted gene expression is an active area of investigation. Diverse signaling pathways converge to induce ZEB1 activity, but few studies have focused on the enzyme-dependent modifications of ZEB1 that occur after its translation from mRNA (i.e., post-translational modifications, PTM). In addition to outlining the current knowledge in the field, we outline several questions regarding the PTM-mediated regulation of ZEB1 that remain unanswered. Thus, the areas of research covered in this review paper will provide a noteworthy conceptual advancement in our understanding of ZEB1′s biological function, as well as its post-transcriptional and post-translational regulation. Furthermore, the review will enhance the development of treatment strategies by identifying knowledge gaps in ZEB1′s regulatory mechanisms that could potentially be targeted to prevent and treat metastasis in cancer patients. Zinc finger E-box binding homeobox 1 (ZEB1) is a pleiotropic transcription factor frequently expressed in carcinomas. ZEB1 orchestrates the transcription of genes in the control of several key developmental processes and tumor metastasis via the epithelial-to-mesenchymal transition (EMT). The biological function of ZEB1 is regulated through pathways that influence its transcription and post-transcriptional mechanisms. Diverse signaling pathways converge to induce ZEB1 activity; however, only a few studies have focused on the molecular associations or functional changes of ZEB1 by post-translational modifications (PTMs). Due to the robust effect of ZEB1 as a transcription repressor of epithelial genes during EMT, the contribution of PTMs in the regulation of ZEB1-targeted gene expression is an active area of investigation. Herein, we review the pivotal roles that phosphorylation, acetylation, ubiquitination, sumoylation, and other modifications have in regulating the molecular associations and behavior of ZEB1. We also outline several questions regarding the PTM-mediated regulation of ZEB1 that remain unanswered. The areas of research covered in this review are contributing to new treatment strategies for cancer by improving our mechanistic understanding of ZEB1-mediated EMT. [ABSTRACT FROM AUTHOR]

Purpose: The mechanism underlying cancer heterogeneity and plasticity remains elusive, in spite of the fact that multiple hypotheses have been put forward. We intended to clarify this heterogeneity in uveal melanoma (UM) by looking for evidence of cancer stem cell involvement and a potential role of ZEB1 in cancer cell plasticity. Methods: Spheroids derived from human UM cells as well as xenograft tumors in nude mice were dissected for signs of heterogeneity and plasticity. Two human UM cell lines were studied: the epithelioid type C918 cell line and the spindle type OCM1 cell line. We knocked down ZEB1 in both cell lines to investigate its involvement in the regulation of stem-like cell formation and vascularization by qRT-PCR, immunohistochemistry, flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. Results: We found that a small side population (SP) in OCM1 showed stem cell-like properties such as heterogeneity, remote dissemination and nuclear dye exclusion after spheroid formation in vitro. ZEB1 regulated UM stem cell generation indirectly by promoting cell proliferation to form large size tumors in vivo and spheroid in vitro, and directly by binding to stemness genes such as TERT and ABCB1. In addition, we found that ZEB1 participates in vasculogenic mimicry system formation through the regulation of CD34 and VE-cadherin expression. Conclusions: From our data we conclude that cancer stem cells may contribute to UM heterogeneity and plasticity and that ZEB1 may play a regulatory role in it. [ABSTRACT FROM AUTHOR]

Purpose of the study. Evaluation of expression of the epithelial-mesenchymal transition markers E-cadherin and ZEB1 in patients with stage II-IV colorectal cancer (CRC).Materials and methods. The study included operational material obtained from 299 patients aged 42–86 years (mean age 64.2±1.7 years) with stage II-IV CRC treated at National Medical Research Centre for Oncology in 2013-2017. Stage II CRC (T3-4 N0 M0 ) was diagnosed in 110 patients, stage III (T1-4 N1-2 M0 ) – in 88 patients, stage IV (T1-4 N0-2 M1 ) – in 101 patients. Polyclonal rabbit antibodies to ZEB1 (Biorbyt Ltd., UK) and mouse monoclonal antibodies to E-cadherin (Diagnostic BioSystems, USA) were used for an IHC analysis. The intensity and degree of tumor cell staining, percentage of stained tumor cells in the sample and the number of patients with positive and negative marker expression were determined. Groups were compared using the Mann–Whitney U test and the Pearson's chi-square test.Results. Positive expression of E-cadherin was found in 64.5 % (193 of 299 patients), ZEB1 – in 80.6 % (241 of 299 patients). The number of patients with E-cadherin-positive tumors statistically significantly decreased (χ2 =15.888 at p

Abstract Background Zinc‐finger E‐box‐binding homeobox 1 (ZEB1) is an important regulator of epithelial‐mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR‐200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT‐mediated acquired resistance to gefitinib in EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. Methods PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as “gefitinib‐resistant persisters” (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. Results GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR‐200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR‐200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133‐ and BMI1‐positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR‐mutant NSCLC patients with gefitinib resistance. Conclusions ZEB1 plays an important role in gefitinib‐resistant lung CSCs with EMT features via regulation of miR‐200c and BMI1.

Abstract 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.

The main aim of the study is to explore the mechanism of microrNA-145-3p targeting ZEB1 on migration and invasion in cervical cancer cells. In this study, cervical cancer cell line C33A was selected and transfected. After transfection of C33A cells, the expression level of Mir-145-3p was measured by QT-PCR. Human cervical cancer cell line (C33A) was cultured in RPMI 1640 medium and cell transfection experiment was performed. The related targets of Mir-145-3p, and a complementary binding site was found between the 3′UTR terminal of ZEB1 and Mir-145-3p was performed using a Targetscan biological software. The luciferase reporter system was used to determine the targeting relationship between Mir-145-3p and complementary binding site. The expression of e-cadherin and vimentin proteins was detected by western blot analysis. After 48 h, the expression level of Mir-145-3p in mimics group was significantly increased, the inhibitor group was significantly down-regulated and was statistically significant (P 0.05), and luciferase activity of Mir-145-3p WT was significantly down-regulated (P > 0.05). Overexpression of microrNA-145-3p downregulated ZEB1 expression and inhibited proliferation, migration, invasion, and epithelial-mesenchymal transformation of cervical cancer cells, and promoted cell apoptosis.

Guanzhang Li,1,* Ting-Wei Chen,2,* Ann-Christin Nickel,2 Sajjad Muhammad,2 Hans-Jakob Steiger,2 Theophilos Tzaridis,3,4 Daniel Hänggi,2 Reinhard Zeidler,5 Wei Zhang,6– 8 Ulf Dietrich Kahlert1,2 1Department of Molecular Neuropathology, Beijing Neurosurgical Institute, Capital Medical University, Beijing, People’s Republic of China; 2Clinic for Neurosurgery, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany; 3Division of Clinical Neurooncology, Department of Neurology and Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, 53127, Germany; 4Tumor Initiation & Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, 92037, USA; 5Department for Otorhinolaryngology, Klinikum der Universität München (LMU), Munich, Germany; 6Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, People’s Republic of China; 7China National Clinical Research Center for Neurological Diseases, Beijing, People’s Republic of China; 8Chinese Glioma Genome Atlas Network (CGGA) and Asian Glioma Genome Atlas Network (AGGA), Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ulf Dietrich Kahlert Moorenstrasse 5, Düsseldorf, 40225, GermanyTel +49-211-810 8013Fax +49-211-810 8012Email mail@ulf-kahlert.comBackground: The metabolic enzyme carbonic anhydrase 12 (CA12/CAXII) emerges as a promising cancer therapeutic target with drug development projects underway. Previous reports proposed the relevance of CA12 in the context of glioma but are limited in patient data quantity, ignore ethnic diversity of patients or rely on semi-quantitative, thereby out of date, methodology. Moreover, little is known on the association of CA12 to brain tumor stemness or on the effect of anti-CAXII-directed monotherapies on glioma stem cells (GSCs), in particular their response regarding mesenchymal differentiation status.Methods: We performed in silico analysis on three independent, large-scale patient datasets interrogating state of the art molecular diagnostics alongside clinical outcomes. We analyzed CAXII abundance on a collection of GSCs and functionally tested their response to exposure to CAXII blocking antibody 6A10.Results: CA12 is highly expressed in glial tumors compared with normal tissue and predicts for poor clinical course of tumor patients. CA12 expression in glioblastoma significantly correlates with clinically established, molecular markers of IDH1WT DNA, WHO grade IV or absence of 1p/19q chromosome arm co-deletion. Furthermore, tumors with elevated CA12 cluster into the mesenchymal transcription subclass of the disease. CAXII abundance in different GSCs ranges from almost absent to high levels and does not correlate to stem cell marker CD133/AC133 cell surface expression. Moreover, aiming to pharmacologically block CAXII in our cells with antibody 6A10 caused significant functional response only in one of the tested GSCs models, featuring suppression of cell invasion accompanied by reduction of ZEB1 protein and other stem cell markers.Conclusion: CA12 represents a clinically relevant and molecular brain tumor-subtype specific therapeutic target. Our correlative data from experimental and clinical samples does not support CA12/CAXII to be GSC specific. 6A10 possesses promising potential to impede the invasive capacity of glioma cells and supports the emerging concept that CAXII interacts with cancer EMT programs. However, further mechanistic studies are required to comprehensively assess the therapeutic potential of 6A10 and to identify different resistance mechanisms of GSCs.Keywords: carbonic anhydrase 12, 6A10, glioma, therapeutic antibody, ZEB1, disease modeling

Ovarian cancer (OvCa) is characterized by early metastasis and high mortality rates, underscoring the need for deeper understanding of these aspects. This study explores the role of glucose transporter 3 (GLUT3) driven by zinc finger E -box-binding homeobox 1 (ZEB1) in OvCa progression and metastasis. Specifically, this study explored whether ZEB1 promotes glycolysis and assessed the potential involvement of GLUT3 in this process in OvCa cells. Our findings revealed that ZEB1 and GLUT3 were excessively expressed and closely correlated in OvCa. Mechanistically, ZEB1 activates the transcription of GLUT3 by binding to its promoter region. Increased expression of GLUT3 driven by ZEB1 dramatically enhances glycolysis, and thus fuels Warburg Effect to promote OvCa progression and metastasis. Consistently, elevated ZEB1 and GLUT3 expression in clinical OvCa is correlated with poor prognosis, reinforcing the profound contribution of ZEB1-GLUT3 axis to OvCa. These results suggest that activation of GLUT3 expression by ZEB1 is crucial for the proliferation and metastasis of OvCa via fueling glycolysis, shedding new light on OvCa treatment. [Display omitted] • Unveiling a novel ZEB1-GLUT3 axis driving ovarian cancer metabolic reprogramming • ZEB1 and GLUT3 levels correlate with poor prognosis in ovarian cancer. • Demonstrated ZEB1-GLUT3 axis' role in promoting ovarian cancer proliferation, migration, and metastasis. [ABSTRACT FROM AUTHOR]

Peiminine, the primary biologically active compound from Fritillaria thunbergii Miq., has demonstrated significant pharmacological activities. Doxorubicin is one of the most potent chemotherapeutic agents for breast cancer (BC). This study was designed to investigate the efficacy and underlying mechanisms of Peiminine combined with Doxorubicin in treating BC. Our results demonstrated that the combination of Peiminine and 1 mg/kg Doxorubicin exhibited more significant suppression of tumor growth compared with the monotherapy in MDA-MB-231 xenograft nude mice model, which is comparable to the effect of 3 mg/kg Doxorubicin in vivo. Notably, the 3 mg/kg Doxorubicin monotherapy resulted in organ toxicity, specifically in the liver and heart, whereas no toxicity was observed in the combination group. In vitro , this combined treatment exhibited a synergistic reduction on the viability of BC cells. Peiminine enhanced the cell cycle arrest and DNA damage induced by Doxorubicin. Furthermore, the combination treatment effectively blocked DNA repair by inhibiting the MAPKs signaling pathways. And ZEB1 knockdown attenuated the combined effect of Peiminine and Doxorubicin on cell viability and DNA damage. In conclusion, our study found that the combination of Peiminine and Doxorubicin showed synergistic inhibitory effects on BC both in vivo and in vitro through enhancing Doxorubicin-induced DNA damage. These findings support that their combination is a novel and promising therapeutic strategy for treating BC. [Display omitted] • Peiminine and Doxorubicin exhibited a synergistic reduction on the viability of breast cancer cells. • The combination of Peiminine and Doxorubicin significantly suppressed tumor growth with no cardiotoxicity. • The synergistic mechanism of Peiminine and Doxorubicin may be related to enhancing DNA damage via downregulation of ZEB1. [ABSTRACT FROM AUTHOR]

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

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

The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I–IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Purpose: The Numb protein plays a vital role in tumor development. The main aim of this study was to identify ATP11A, which is associated with the biological behavior of pancreatic cancer, and elucidate its relationship with Numb and the underlying mechanism behind this relationship. Methods: First, data retrieved from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEX) databases was used to investigate the expression of ATP11A mRNA and its relationship with Numb mRNA in pancreatic cancer. Western blot assays on 31 pairs of pancreatic cancer tissues and paracancerous tissues, and immunohistochemical assays on 81 pancreatic cancer specimens were performed in order to verify the expression of ATP11A in pancreatic cancer at the protein level. Next, ATP11A was overexpressed or knocked down to observe its effects on the invasion and migration ability of pancreatic cancer cells and the changes of downstream proteins. Rescue assays were conducted to determine the mechanism through which ATP11A affects Numb, ZEB1, Snail2 and other proteins. Furthermore, immunoprecipitation assays were performed to explore the interaction between ATP11A and Numb. Finally, pancreatic cancer cells were stimulated with TGFB1 and ATP11A expression was examined to explore whether the effect of ATP11A on EMT was TGFB dependent. Results: At the mRNA level, the expression of ATP11A in pancreatic cancer tissues was significantly higher than in normal pancreatic tissues (P < 0.001). ATP11A expression was also highly correlated with Numb expression (R = 0.676). At the protein level, ATP11A expression in pancreatic cancer tissues was significantly higher than that in paracancerous tissues (P = 0.0009), and high ATP11A expression was also correlated with a worse prognosis. Moreover, our results showed that ATP11A can promote the invasion and migration of pancreatic cancer cells. Additionally, ATP11A could positively regulate the expression of Numb PRRL, Snail2 and ZEB1 proteins. The rescue experiment results showed that the enhancement effect of ATP11A on ZEB1/Snail2 was suppressed by the specific knockdown of Numb PRRL. In addition, the immunoprecipitation results showed that ATP11A could specifically bind to Numb PRRL. The expression of ATP11A was also upregulated after TGFB stimulation, suggesting that the effect of ATP11A on EMT is TGFB dependent. Conclusion: ATP11A is significantly upregulated in pancreatic cancer tissues, where it promotes the invasion and migration ability of pancreatic cancer cells. It is also associated with adverse prognosis in pancreatic cancer. Furthermore, ATP11A affects the epithelial-to-mesenchymal transition (EMT) of pancreatic cancer by regulating the TGFB dependent Numb PRRL-ZEB1/Snail2 pathway. [ABSTRACT FROM AUTHOR]

Cardiac mesenchymal cells (CMCs) are a promising cell type that showed therapeutic potential in heart failure models. The analysis of the underlying mechanisms by which the CMCs improve cardiac function is on track. This study aimed to investigate the expression of N‐Cadherin, a transmembrane protein that enhances cell adhesion, and recently gained attention for differentiation and augmentation of stem cell function. The mouse CMCs were isolated and analyzed for the mesenchymal markers using flow cytometry. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis were used to assess the expression of N‐Cadherin along with its counteracting molecule E‐Cadherin and their regulator Zeb1 in CMCs and dermal fibroblast. The expression level of miR‐200c and miR‐429 was analyzed using miRNA assays. Transient transfection of miR‐200c followed by qRT‐PCR, western blot analysis, and immunostaining was done in CMCs to analyze the expression of Zeb1, N‐Cadherin, and E‐Cadherin. Flow cytometry analysis showed that CMCs possess mesenchymal markers and absence for hematopoietic and immune cell markers. Increased expression of N‐Cadherin and Zeb1 in CMCs was observed in CMCs at both RNA and protein levels compared to fibroblast. We found significant downregulation of miR‐200c and miR‐429 in CMCs. The ectopic expression of miR‐200c in CMCs significantly downregulated Zeb1 and N‐Cadherin expression. Our findings suggest that the significant downregulation of miR‐200c/429 in CMCs maintains the expression of N‐Cadherin, which may be important for its functional integrity. [ABSTRACT FROM AUTHOR]

Pancreatic cancer (PC) often correlates with high mortality due to late diagnosis, rapid metastasis, and resistance to chemotherapy. miR-128-3p has been validated as a tumor suppressor in PC. This study explored the functional mechanism of miR-128-3p in epithelial-mesenchymal transition (EMT) of PC cells. Four PC cancer cell lines with different degrees of malignancy and normal pancreatic cells were selected to detect expressions of hsa-miR-128-3p and ZEB1 by RT-qPCR and Western blot. miR-128-3p mimic or si-ZEB1 was delivered into PANC-1 cells and miR-128-3p inhibitor or oe-ZEB1 was delivered into AsPC-1 cells. Expressions of epithelial and mesenchymal markers were analyzed by Western blot and cell fluorescence staining. The binding relationship between miR-128-3p and ZEB1 was examined by bioinformatics analysis and dual-luciferase assay, and verified by RT-qPCR and Western blot. PC cell invasion and migration were assessed by Transwell assays. Generally, hsa-miR-128-3p was poorly-expressed in PC cells. However, it was relatively more expressed in AsPC-1 cells with epithelial phenotypes relative to PANC-1 cells with mesenchymal phenotype, whereas ZEB1 expression showed opposite tendencies. PANC-1 cells transfected with miR-128-3p mimic or si-ZEB1 showed upregulated E-cadherin and downregulated N-cadherin, and transformed from mesenchymal phenotypes to epithelial phenotypes, with decreased invasion and migration, while opposite results occurred in AsPC-1 cells transfected with miR-128-3p inhibitor or oe-ZEB1. miR-128-3p targeted ZEB1. oe-ZEB1 antagonized the inhibition of miR-128-3p mimic on PANC-1 cell EMT, invasion, and migration, while si-ZEB1 reversed the facilitation of miR-128-3p inhibitor in AsPC-1 cells. In conclusion, miR-128-3p inhibited PC cell EMT, invasion, and migration by targeting ZEB1. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells. [Display omitted] • Zeb1 facilitates autophagy likely through the regulation of autophagic genes. • Zeb1 regulates autophagy independently of p53 status. • Zeb1 augments resistance of the breast cancer cells to genotoxic drugs, at least partially, via autophagy. [ABSTRACT FROM AUTHOR]

Noncoding circular RNAs (circRNAs) have participated in the progression of preeclampsia (PE) via inhibiting microRNAs (miRNAs) to regulate gene expression. This study was designed to explore the miRNA/mRNA mechanism of hsa_circ_0007121 (circ_0007121) in PE. The expression detection of circ_0007121, microRNA-421 (miR-421), and zinc finger E-box binding homeobox 1 (ZEB1) was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay. Transwell assay was used to determine cell migration and invasion. Cell apoptosis was evaluated using flow cytometry. The protein levels of epithelial-mesenchymal transition (EMT) markers and ZEB1 were measured via western blot. The interaction between miR-421 and circ_0007121 or ZEB1 was validated by dual-luciferase reporter assay. The expression detection indicated that circ_0007121 was downregulated in PE patients and the clinical data revealed that circ_0007121 was related to PE. The upregulation of circ_0007121 promoted cell proliferation, migration, invasion, and EMT in trophoblastic cells. Furthermore, circ_0007121 was identified as a sponge of miR-421 and the function of circ_0007121 was dependent on the sponge effect on miR-421. Moreover, ZEB1 was a target of miR-421 and circ_0007121/miR-421 axis could regulate the expression of ZEB1. In addition, miR-421 overexpression repressed trophoblastic cell behaviors through downregulating the ZEB1 level. Altogether, circ_0007121 contributed to the development of trophoblastic cells by regulating the miR-421/ZEB1 axis. [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related deaths worldwide. Cisplatin-based chemotherapy remains the standard gold method in therapeutic strategy due to the high usefulness of cisplatin, the application, and the dosage of cisplatin regulated by its adverse effects on neurons. In addition, salicylic acid as a nonsteroidal anti-inflammatory drug has also been reported to treat another type of cancer by activating apoptosis mechanisms. In this research, the synergistic function of cisplatin and salicylic acid on apoptosis activity, cytotoxicity of lung cell lines A549 and Calu-6, and molecular expression level of ZEB1 and P21 genes were the main objectives. Lung carcinoma cell lines A549 and Calu-6 were exposed to different concentrations of cisplatin and salicylic acid alone and in combination to evaluate cell viability by MTT assay. Then, the optimal concentration of cisplatin (0.5 µM) in combination with salicylic acid (2 mM) for apoptotic cells was determined by flow cytometry. Real-time PCR was also performed to conduct the expression analysis of P21 and ZEB1 genes. The combination of the two drugs showed the highest inhibitory and anti-proliferation effects on A549 and Calu-6 cell lines (P ˂ 0.05; P ˂ 0.01, respectively). Gene expression analysis showed that the P21 gene was significantly downregulated in the Calu-6 cell lines and the ZEB1 gene was downregulated only in the A549 cell line (P = 0.01). ZEB1 was overexpressed in the Calu-6. The combined form of cisplatin and salicylic acid significantly suppressed cell proliferation and promoted apoptosis in lung cancer cell lines. [ABSTRACT FROM AUTHOR]

Zinc finger E-box binding homeobox 1 (ZEB1) has been widely recognized as an important driver of tumor growth and metastasis. However, nothing is known about ZEB1-regulated circular (circ)RNAs in cancer. In the current study, we evaluated the function of a novel ZEB1-regulated circRNA derived from the WWC3 gene locus, circWWC3 in breast cancer progression. We found that ZEB1 upregulated circWWC3 expression but not the linear WWC3 mRNA expression. circWWC3 is highly expressed in breast cancer tissues and is associated with the poor prognosis of breast cancer patients. Silencing of circWWC3 significantly suppresses the proliferation, migration, and invasion of breast cancer cells. Mechanically, circWWC3 upregulates multiple oncogenes’ expression of the Ras signaling pathway through acting as the sponge of microRNA (miR)-26b-3p and miR-660-3p. Moreover, short hairpin (sh)RNA-mediated knockdown of circWWC3 partially antagonized ZEB1-mediated breast cancer growth and metastasis in vivo. Our findings reveal that ZEB1-mediated upregulation of circWWC3 promotes breast cancer progression through activating Ras signaling pathway, which provides a potential therapeutic target and prognostic biomarker for breast cancer.

Jinrui Sun,1 Shanshan Wang,2 Ping Liu,1 Yulan Liu3 1Department of Gynecology, Shanxi Provincial People’s Hospital, Taiyuan, Shanxi Province 030012, People’s Republic of China; 2Department of Cardiology, Yidu Central Hospital of Weifang City, Weifang, Shandong, People’s Republic of China; 3Department of Gynecology, Maternal and Child Health Hospital of Hubei Province, Wuhan, Hubei 430070, People’s Republic of ChinaCorrespondence: Yulan LiuDepartment of Gynecology, Maternal and Child Health Hospital of Hubei Province, NO. 745 Wuluo Road, Hongshan, Wuhan, Hubei 430070, People’s Republic of ChinaTel +86-15698784852Email yulantang885@163.comObjective: To verify that miR-139-5p-zinc finger E-box-binding homeobox 1 (ZEB1) is a molecular regulator of the biological function and epithelial–mesenchymal transition (EMT) of cervical cancer (CC) cells.Methods: Cancerous tissues, corresponding paracancerous tissues, and serum were sampled from patients with CC. MiR-139-5p and ZEB1 in tissue specimens, serum specimens, and purchased CC cell lines were quantified, and Pearson correlation coefficient was adopted for correlation analysis of miR-139-5p in clinical specimens. Receiver operating characteristic (ROC) curves were adopted to analyze the diagnostic value of miR-139-5p and ZEB1 for CC. The expression of genes in CC cells was changed by transfection. The proliferation, colony formation, invasion, and apoptosis of cells were determined, and the protein level of EMT markers (N-cadherin, vimentin, and E-cadherin) was also quantified. Moreover, the targeting relationship between miR-139-5p and ZEB1 was determined.Results: Our data showed that the expression of miR-139-5p decreased greatly in CC tissues, and it also significantly decreased in the serum, while the expression of serum ZEB1 was opposite. In addition, the miR-139-5p expression in CC tissues was positively correlated with that in serum, while serum miR-139-5p was negatively correlated with serum ZEB1. The areas under the curves (AUCs) of the two for identifying CC were 0.923 and 0.890, respectively. Both up-regulation of miR-139-5p and down-regulation of ZEB1 suppressed the colony formation, proliferation, invasion, and EMT of CC cells, and intensified their apoptosis. Moreover, miR-139-5p negatively regulated the transcription of ZEB1, and down-regulation of the former could reverse the molecular regulatory effects of down-regulating ZEB1 on the above biological behaviors of CC cells.Conclusion: The above data imply that miR-139-5p-ZEB1 axis may be the key to curbing the progression of CC.Keywords: cervical cancer, miR-139-5p, ZEB1, growth, invasion, epithelial–mesenchymal transition

Abstract Background Bladder cancer (BC) is a common urinary neoplasm with high incidence worldwide. Long noncoding RNA zinc ribbon domain containing 1 antisense RNA 1 (ZNRD1‐AS1) has been reported to be upregulated in BC. However, the exact role of ZNRD1‐AS1 as well as its mechanism remains poorly understood. Methods Zinc ribbon domain containing 1 antisense RNA 1, and its potential downstream genes microRNA‐194 (miR‐194) and zinc finger E‐box binding homeobox 1 (ZEB1) levels were detected via quantitative real‐time polymerase chain reaction or western blot. Cell proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) were detected to assess the influences of ZNRD1‐AS1, miR‐194 and ZEB1 on BC cells by colony formation, cell counting kit‐8 (CCK‐8), transwell analysis or western blot. The relationship between miR‐194 and ZNRD1‐AS1 or ZEB1 was analyzed by luciferase activity analysis. The xenograft experiment was performed to assess the function of ZNRD1‐AS1 in vivo. Results Zinc ribbon domain containing 1 antisense RNA 1level was upregulated in BC. ZNRD1‐AS1 silence repressed proliferation, migration, invasion and EMT in BC cells. MiR‐194 was identified as a target of ZNRD1‐AS1, and miR‐194 upregulation repressed proliferation, migration, invasion, and EMT by ZNRD1‐AS1 sponging. ZEB1 was targeted via miR‐194 and its interference impeded proliferation, migration, invasion, and EMT. Moreover, ZNRD1‐AS1 regulated ZEB1 expression via miR‐194. Besides, inhibition of ZNRD1‐AS1 attenuated tumor growth by miR‐194/ZEB1 axis in vivo. Conclusion Knockdown of ZNRD1‐AS1 suppressed BC cell development in vitro and in vivo via targeting miR‐194 to regulate ZEB1, indicating a novel avenue for treatment of BC.

Purpose The Numb protein plays a vital role in tumor development. The main aim of this study was to identify ATP11A, which is associated with the biological behavior of pancreatic cancer, and elucidate its relationship with Numb and the underlying mechanism behind this relationship. Methods First, data retrieved from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEX) databases was used to investigate the expression of ATP11A mRNA and its relationship with Numb mRNA in pancreatic cancer. Western blot assays on 31 pairs of pancreatic cancer tissues and paracancerous tissues, and immunohistochemical assays on 81 pancreatic cancer specimens were performed in order to verify the expression of ATP11A in pancreatic cancer at the protein level. Next, ATP11A was overexpressed or knocked down to observe its effects on the invasion and migration ability of pancreatic cancer cells and the changes of downstream proteins. Rescue assays were conducted to determine the mechanism through which ATP11A affects Numb, ZEB1, Snail2 and other proteins. Furthermore, immunoprecipitation assays were performed to explore the interaction between ATP11A and Numb. Finally, pancreatic cancer cells were stimulated with TGFB1 and ATP11A expression was examined to explore whether the effect of ATP11A on EMT was TGFB dependent. Results At the mRNA level, the expression of ATP11A in pancreatic cancer tissues was significantly higher than in normal pancreatic tissues (P < 0.001). ATP11A expression was also highly correlated with Numb expression (R = 0.676). At the protein level, ATP11A expression in pancreatic cancer tissues was significantly higher than that in paracancerous tissues (P = 0.0009), and high ATP11A expression was also correlated with a worse prognosis. Moreover, our results showed that ATP11A can promote the invasion and migration of pancreatic cancer cells. Additionally, ATP11A could positively regulate the expression of Numb PRRL, Snail2 and ZEB1 proteins. The rescue experiment results showed that the enhancement effect of ATP11A on ZEB1/Snail2 was suppressed by the specific knockdown of Numb PRRL. In addition, the immunoprecipitation results showed that ATP11A could specifically bind to Numb PRRL. The expression of ATP11A was also upregulated after TGFB stimulation, suggesting that the effect of ATP11A on EMT is TGFB dependent. Conclusion ATP11A is significantly upregulated in pancreatic cancer tissues, where it promotes the invasion and migration ability of pancreatic cancer cells. It is also associated with adverse prognosis in pancreatic cancer. Furthermore, ATP11A affects the epithelial-to-mesenchymal transition (EMT) of pancreatic cancer by regulating the TGFB dependent Numb PRRL-ZEB1/Snail2 pathway.

Metformin is an important antidiabetic drug that has the potential to reduce skeletal muscle atrophy and promote the differentiation of muscle cells. However, the exact molecular mechanism underlying these functions remains unclear. Previous studies revealed that the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), which participates in tumor progression, inhibits muscle atrophy. Therefore, we hypothesized that the protective effect of metformin might be related to ZEB1. We investigated the positive effect of metformin on IL-1β-induced skeletal muscle atrophy by regulating ZEB1 in vitro and in vivo. Compared with the normal cell differentiation group, the metformin-treated group presented increased myotube diameters and reduced expression levels of atrophy-marker proteins. Moreover, muscle cell differentiation was hindered, when we artificially interfered with ZEB1 expression in mouse skeletal myoblast (C2C12) cells via ZEB1-specific small interfering RNA (si-ZEB1). In response to inflammatory stimulation, metformin treatment increased the expression levels of ZEB1 and three differentiation proteins, MHC, MyoD, and myogenin, whereas si-ZEB1 partially counteracted these effects. Moreover, marked atrophy was induced in a mouse model via the administration of lipopolysaccharide (LPS) to the skeletal muscles of the lower limbs. Over a 4-week period of intragastric administration, metformin treatment ameliorated muscle atrophy and increased the expression levels of ZEB1. Metformin treatment partially alleviated muscle atrophy and stimulated differentiation. Overall, our findings may provide a better understanding of the mechanism underlying the effects of metformin treatment on skeletal muscle atrophy and suggest the potential of metformin as a therapeutic drug., (Copyright © 2024 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Objective: Sex-determining region-Y-related high-mobility-group box 4 (SOX4) is associated with the metastasis and prognosis of many cancer types. However, studies on the role of SOX4 in laryngeal squamous cell carcinoma (LSCC) are few, and hence the mechanism is unclear. Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the plasticity and motility required for tumor progression and metastasis. This study aimed to analyze the relationship between SOX4 and EMT, and their relationship with clinicopathological factors and related prognosis.Methods: Immunohistochemical staining was used to detect the positive expression of SOX4 protein, EMT-related transcription factor protein, and related marker protein in 127 LSCC tissue samples. At the same time, data on various parameters of clinical pathology and postoperative survival were collected.Results: The positive expression rate of SOX4 and Slug in LSCC was related to pathological differentiation, Lymph node metastasis (LNM), and pathological TNM of a tumor. The expression rates of ZEB1, E-cadherin, N-cadherin, and β-catenin in LSCC correlated with LNM and pTNM. The expression of SOX4, combined expression of SOX4 and ZEB1, and LNM were independent prognostic factors for the total survival time of patients with LSCC.Conclusions: In summary, SOX4 was vital in the LSCC EMT process, which might be mediated by transcription factor ZEB1. SOX4 and ZEB1 might serve as potential biomarkers of metastasis and prognosis, as well as promising therapeutic targets of LSCC. [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: Many studies have reported the roles of the extracellular hypoxia microenvironment in the tumorigenesis and metastasis of multiple cancers. However, long noncoding RNAs (lncRNA s) that induce cancer oncogenicity and metastasis of pancreatic cancer (PC) under hypoxia conditions remain unclear. Methods: In PC cells, the expression levels of lncRNA s in different conditions (normoxia or hypoxia) were compared through RNA sequencing (RNA-seq). The effects of the zinc finger E-box-binding homeobox 1 (ZEB1-AS1) antisense lncRNA on PC cells cultured in normoxia/hypoxia medium were measured through gain and loss-of-function experiments. Fluorescence in situ hybridization and luciferase reporter assays in addition to in vivo studies were utilized to explore the adaptive mechanisms of ZEB1-AS1 in the hypoxia-promoted proliferation, migration, and invasion ability of PC cells. Moreover, the level of ZEB1-AS1 and its associated targets or pathways were investigated in both PC and pancreatic normal tissues. Results: RNA-seq revealed that ZEB1-AS1 was significantly upregulated in PC cells under hypoxia conditions. The ZEB1-AS1 expression level was closely associated with poor prognosis of PC patients. Knockdown of ZEB1-AS1 suppressed the proliferation, migration, and invasion of PC cells in vitro as well as PC xenograft tumor growth in vivo. In PC cells, RNAi-mediated reduction of ZEB1-AS1 inhibited zinc finger E-box-binding homeobox 1 (ZEB1), while ZEB1-AS1 overexpression rescued ZEB1 expression, indicating that ZEB1-AS1 promotes ZEB1 expression. Moreover, hypoxia-inducible factor-1α (HIF-1 α)induced the expression of ZEB1-AS1 by binding to the ZEB1-AS1 promoter, which contains a putative hypoxia response element (HRE). Mechanistically, ZEB1-AS1 scaffolded the interaction among HIF-1 α, ZEB1 , and histone deacetylase 1 (HDAC1), leading to deacetylation-mediated stabilization of HIF-1 α. We further revealed that ZEB1 induced the deacetylase capacity of HDAC1 to suppress the acetylation or degradation of HIF-1 α, improving HIF-1 α assembly. Thus, hypoxia-induced ZEB1-AS1 facilitated ZEB1 transcription and the stability of HIF-1 α, which promoted the metastasis of PC cells. Clinically, dysregulated ZEB1 and HIF-1 α expression was significantly correlated with histological grade, lymphatic metastasis, and distant metastasis in PC patients. Conclusions: Our results emphasized that the positive reciprocal loop of HIF-1 α/ ZEB1-AS1 / ZEB1 / HDAC1 contributes to hypoxia-promoted oncogenicity and PC metastasis, indicating that it might be a novel therapeutic target for PC. [ABSTRACT FROM AUTHOR]

The transcription factor Zinc finger E-box binding 1 (ZEB1) displays a range of regulatory activities in cell function and embryonic development, including driving epithelial-mesenchymal transition. Several aspects of ZEB1 function can be regulated by its functional interactions with noncoding RNA types, namely microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Increasing evidence indicates that ZEB1 importantly influences cancer initiation, tumor progression, metastasis, and resistance to treatment. Cancer is the main disease-related cause of death in children and adolescents. Although the role of ZEB1 in pediatric cancer is still poorly understood, emerging findings have shown that it is expressed and regulates childhood solid tumors including osteosarcoma, retinoblastoma, neuroblastoma, and central nervous system tumors. Here, we review the evidence supporting a role for ZEB1, and its interplays with miRNAs and lncRNAs, in pediatric cancers. [ABSTRACT FROM AUTHOR]

ZEB1 is an important transcription factor that plays a critical role in TGF-β-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the mechanisms by which ZEB1 regulates metastasis in esophageal squamous cell carcinoma (ESCC) remain largely unknown. Here, we identified a novel circular RNA, circ-DOCK5, the biogenesis of which is directly regulated by ZEB1 and ZEB1-repressed RNA-binding protein eIF4A3. Tissue microarray analysis identified circ-DOCK5 to be downregulated in ESCC tissues, and its downregulation correlated with poor prognosis. Moreover, circ-DOCK5 increased the stability of miR-627-3p by functioning as a "reservoir" for miR-627-3p to partially reverse the ZEB1-enhanced migration and invasion in ESCC. MiR-627-3p inhibited the expression of TGFB2 and the secretion of TGF-β, which further resulted in downregulation of ZEB1 and suppression of TGF-β-induced EMT. In vivo experiments showed that ZEB1 promoted metastasis in ESCC by regulating expression of circ-DOCK5. Therefore, the present study revealed that ZEB1-mediated downregulation of circ-DOCK5 facilitates metastasis in ESCC by forming a positive feedback loop with TGF-β by altering the miR-627-3p/TGFB2 signaling. Targeting this signaling pathway may help suppress progression in ESCC. [ABSTRACT FROM AUTHOR]

DEPTOR plays vital roles in the regulation of cell proliferation and survival by directly modulating the activity of mTORC1/2. However, the physiological role of DEPTOR in lung tumorigenesis, as well as its clinical significance, remains elusive. In this study, we revealed that decreased DEPTOR expression correlated with increased tumor size, poor differentiation, and worse survival in patients with lung cancer. DEPTOR depletion promoted cell proliferation, survival, migration, and invasion in human lung cancer cells. Mechanistically, DEPTOR bound to the kinase domain of EGFR via its PDZ domain to inactivate EGFR signal. Thus, DEPTOR depletion not only directly activated mTORC1/2, but also relieved the inhibition of EGFR to subsequently activate mTOR signals, leading to the induction of cell proliferation and survival. Additionally, activated EGFR-mTOR signals upregulated the expression of ZEB1 and SLUG to induce epithelial-mesenchymal transition, resulting in enhanced migration and invasion. Importantly, Deptor deletion accelerated KrasG12D;p53fl/fl-induced lung tumorigenesis and shortened mouse life span via the activation of EGFR-mTOR signals. Collectively, our study demonstrated that DEPTOR acts as a tumor suppressor in lung tumorigenesis, and its reduction may advance the progression of human lung cancer. [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]

Long noncoding RNA (lncRNA) have emerged as crucial regulators for a myriad of biological processes, and perturbations in their cellular expression levels have often been associated with cancer pathogenesis. In this study, we identified AATBC (apoptosis‐associated transcript in bladder cancer, LOC284837) as a novel lncRNA. AATBC was found to be highly expressed in nasopharyngeal carcinoma (NPC), and increased AATBC expression was associated with poor survival in patients with NPC. Furthermore, AATBC promoted migration and invasion of NPC cells in vitro, as well as metastasis in vivo. AATBC upregulated the expression of the desmosome‐associated protein pinin (PNN) through miR‐1237‐3p sponging. In turn, PNN interacted with the epithelial–mesenchymal transition (EMT) activator ZEB1 and upregulated ZEB1 expression to promote EMT in NPC cells. Collectively, our results indicate that AATBC promotes NPC progression through the miR‐1237‐3p–PNN–ZEB1 axis. Our findings indicate AATBC as a potential prognostic biomarker or therapeutic target in NPC.

Kai Wu,1,* Qiong Feng,2,* Liang Li,1 Yanfei Xiong,3 Shihong Liu,4 Jie Liu,1 Qing Wu1 1Department of Orthopedics, The Second Affiliated Hospital of Nanchang University, Nanchang 300006, People’s Republic of China; 2Nursing School, Nanchang University, Nanchang 300006, People’s Republic of China; 3Department of Orthopedics, Jingan Hospital, Yichun 330600, People’s Republic of China; 4Jingan Hospital of Traditional Chinese Medicine, Yichun 330600, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qing Wu Email wuqing18186@126.comIntroduction: The long-noncoding RNA PCAT6 plays an important regulatory role in the development of several cancers. However, the expression pattern and underlying mechanisms of PCAT6 in osteosarcoma (OS) are yet unknown.Methods: We used real-time PCR to measure PCAT6 expression in 106 tumor pairs and corresponding non-tumor tissues from OS patients. Statistical analyses were applied to evaluate the prognostic value and associations of PCAT6 expression with clinical parameters. Furthermore, the PCAT6 was silenced with siRNA in OS cells. Moreover, phenotype of PCAT6 silenced OS cells was measured using colony formation, CCK-8, cell migration and invasion assay. Finally, the molecular mechanism of PCAT6/miR-143-3p/ZEB1 axis in OS progression was explored.Results: The expression level of PCAT6 in OS tissues was significantly elevated as compared with that in the adjacent normal bone tissues and that high PCAT6 expression closely correlated with the malignant phenotype and poor survival among patients with OS. Multivariate analyses revealed PCAT6 overexpression as an independent prognostic factor for the poor outcome of patients with OS. Functional assay results demonstrated that the knockdown of PCAT6 expression notably suppressed the proliferation, migration, and invasion of OS cells. An elevated PCAT6 level aggravated the malignant phenotype of OS cells via ZEB1 expression upregulation. Mechanistic studies revealed that PCAT6 could sponge endogenous miR-143-3p and inhibit its activity, resulting in an increase in ZEB1 level. Finally, we demonstrated that the tumour-promoting role of PCAT6 in OS was dependent on the regulation of the miR-143-3p/ZEB1 axis.Conclusion: These findings highlight the potential role of PCAT6, which could serve as a valuable prognostic indicator for patients with OS.Keywords: osteosarcoma, PCAT6, ZEB1, miR-143-3p

Objective To investigate the diagnostic value and regulatory mechanism of miR-200a targeting ZEB1 in pregnancy-induced hypertension (PIH). Methods The expression of miR-200a and ZEB1 was detected in the placenta of PIH patients, and then the human trophoblastic cell line JEG-3 was transfected and divided into different groups: control group, NC group, ZEB1 siRNA group, miR-200a inhibitor group and miR-200a inhibitor group + ZEB1 siRNA group. After transfection, cell proliferation and migration/invasion were evaluated byMTT and Transwell assays, respectively, whereas apoptosis was assessed byflow cytometry. MiR-200a was measured by qRT-PCR, while ZEB1 was detectedby Western blotting. Results The expression of miR-200a was gradually increased in the placenta of patients with hypertension and mild or severe preeclampsia, while the mRNA and protein levels of ZEB1 were downregulated. A dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-200a andZEB1.Compared to the control, the miR-200a inhibitor caused a strongdecrease in miR-200a andan upregulation of ZEB1, with a significant enhancement ofcell proliferation, migration and invasion and a decrease in apoptosis. However, no significant alteration was observedin the miR-200a level after administration of ZEB1 siRNA, while ZEB1 was downregulated, with significant suppression of growth. Conclusion MiR-200a was upregulated in PIH patients, andinhibition of miR-200a may improve disease progression, as it could facilitatetrophoblastproliferation, migration and invasionandinhibitapoptosisby targeting ZEB1.

Abstract 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.

Lirui He, Yunpeng Lei, Jianing Hou, Jianlong Wu, Guoqing Lv Department of Gastrointestinal Surgery, Peking University Shenzhen Hospital, Shenzhen, Guangdong 518000, People’s Republic of ChinaCorrespondence: Guoqing LvPeking University Shenzhen Hospital, No. 1120 Lianhua Road, Futian District, Shenzhen, Guangdong 518000, People’s Republic of ChinaEmail GuoqingLv123@126.comBackground: Gastric cancer (GC) is an aggressive malignancy with high lethality. Systematic chemotherapy is the main therapeutic strategy for advanced GC patients. The overexpression of Axl is associated with poor prognosis and regulates tumor growth and metastasis in many types of cancer. However, the role of Axl in GC progression remains elusive.Materials and Methods: Western blot and quantitative real-time PCR assay (RT-PCR) assays were used to detect the expression of Gas6, Axl, ZEB1 and epithelial-mesenchymal transition (EMT)-related markers in GC cells. Cell proliferation was determined by EdU cell proliferation assay and CCK-8 assay. Transwell invasion assay was performed to explore the effect of Axl and ZEB1 on cell invasion. Tumor xenografts and lung metastasis models were conducted to examine the effect of Axl on the growth and lung metastasis of GC cells.Results: In our study, we found that high levels of Gas6 and Axl expression were associated with reduced overall survival (OS) in GC patients and the expression of Gas6 and Axl was upregulated in GC cell lines. Ectopic expression of Axl induced EMT and promoted GC cell invasion and proliferation. The knockdown of Axl inhibited EMT and suppressed the proliferation and invasion of GC cell. In vivo study showed that inhibition of Axl impaired tumor growth and lung metastasis of GC cells. Mechanistic investigations revealed that Axl promoted EMT, invasion, and proliferation via upregulating ZEB1 expression in GC cells.Conclusion: Our results demonstrated that the Gas6/Axl/ZEB1 signaling pathway regulated EMT, invasion, and proliferation in GC cells and might represent a potential therapeutic target for GC treatment.Keywords: gastric cancer, Gas6/Axl signaling pathway, ZEB1, EMT, invasion, proliferation

Objective To explore the expression of ZEB1-AS1 in endometrial cancer (EC), and its correlation with clinicopathological features. Methods The expression of ZEB1-AS1 and ZEB1 in 195 pairs of endometrial cancer and paracancerous tissues were detected by qRT-PCR analysis. We analyzed the correlation of ZEB1-AS1 and ZEB1 expression with the clinical characteristics of EC patients. MTT assay, Transwell migration assay and invasion assay were used to detect the proliferation, migration and invasion of EC cell lines after ZEB1-AS1 overexpression. Results ZEB1-AS1 and ZEB1 were upregulated in EC tissues (P < 0.05). The high expression level of ZEB1-AS1 and ZEB1 were positively correlated and could shorten the overall survival time of EC patients (P < 0.05). The high expression levels of ZEB1-AS1 was correlated with family history, pathology grade, pathological type, chemotherapy, clinical stage, T stage, M stage and HPV infection of EC patients (P < 0.05). The high expression levels of ZEB1 was correlated with family history, pathology grade, clinical stage, T stage, N stage and M stage (P < 0.05). Overexpression of ZEB1-AS1 promoted the proliferation, migration and invasion of EC cell lines (all P < 0.05). Conclusion Both ZEB1-AS1 and ZEB1 are upregulated in EC tissues and could promote the metastasis, invasion of endometrial cancer and indicate the poor prognosis.

Bin Dai, Zhiyong Xiao, Guangtong Zhu, Beibei Mao, Hui Huang, Feng Guan, Zhenyang Lin, Weicheng Peng, Xin Liang, Bolun Zhang, Zhiqiang Hu Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Peking University Ninth School of Clinical Medicine, Beijing, People’s Republic of ChinaCorrespondence: Zhiqiang HuDepartment of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Peking University Ninth School of Clinical Medicine, No. 10 Tieyi Road, Yangfangdian, Haidian District, Beijing 10038, People’s Republic of ChinaTel +86 10 63926627 Email huzhiqbj@sina.comBackground: Glioma is one of the important diseases that threaten human survival in today’s society. WD repeat domain 5 (WDR5) belongs to the components of the lysine methyltransferase complex. WDR5 is involved in gene transcription regulation, cell senescence, cancer and other biological events through methylation modification. However, its expression and function in glioma are still unclear.Materials and Methods: The expression of WDR5 was observed in glioma cells, and then a glioma cell line SW1783 knocked down WDR5 was established. The effects of the decrease of WDR5 expression on proliferation, migration, invasion and EMT of glioma cells were detected, respectively. The downstream regulators of WDR5 were identified by gene expression profiling technology, and the possible molecular mechanisms were identified.Results: In this study, we found that WDR5 could promote glioma cell’s proliferation, migration, invasion and tumor metastasis. In glioma, especially in metastatic glioma tissues, WDR5 levels were significantly increased, the higher expression level could also cause a significant reduction in overall survival of glioma patients. Second, the ability of cells’ proliferation, migration, invasion and tumor metastasis was significantly reduced in WDR5 knockdown cell lines. We also found a significant change in the expression level of epithelial and mesenchymal markers in WDR5 knockdown cell lines. Furthermore, we found that knockdown of WDR5 could inhibit the expression of zinc finger E-box binding homeobox 1 (ZEB1), and knockdown of ZEB1 could inhibit invasion, migration and epithelial–mesenchymal transformation (EMT) in WDR5 over-expression cell line. We also found that WDR5 may regulate ZEB1’s expression through H3K4me3.Conclusion: In summary, in this study, we have studied the relationship between WDR5 and glioma, and found that WDR5’s expression is positively correlated with the proliferation, migration, and invasion of glioma cells, which will help find the potential therapeutic target for glioma patients.Keywords: WDR5, ZEB1, glioma, migration, metastasis

Juan Bai,1 Wen-Yu Jiao2 1Department of Oncology, Affiliated Hospital of Chengdu University, Chengdu 610081, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, Xi’an Daxing Hospital, Xi’an 710016, People’s Republic of ChinaCorrespondence: Wen-Yu Jiao Email xopobs@163.comObjective: MicroRNA-199a-3p (miR-199a-3p or miR-199b-3p) targeting of 3ʹ-UTR of ZEB1 was characterized as an important way to inhibit invasion and metastases in non-small cell lung cancer (NSCLC), one of the most common cancers around the world. Here we aimed to investigate the tumor-suppressive role of miR-199a-3p targeted ZEB1.Materials and Methods: A549 cells were transfected with ZEB1 and/or miR-199a-3p. Then, tumor growth was investigated in xenograft mice. Stem-like property, proliferation and mitochondria injury were further validated in vitro.Results: Overexpression of miR-199a-3p with premiRNAs significantly reduced tumor growth inhibited CD44 and Ki67 and increased Caspase-3 in A549 xenograft mice. Sphere formation and protein expression of stem-like markers showed that miR-199a-3p inhibited stemness of A549 cell. miR-199a-3p reduced proliferation of A549 cells, as showed with EdU staining and reduced expression of Ki67. Transfection of miR-199a-3p also promoted apoptosis, as indicated with increased apoptotic cells with flow cytometry, and increased cleaved Caspase-3/Caspase3 and Bcl-2/Bax. Apoptosis was further validated to be induced with mitochondria dysfunction, which indicated with JC-1 labeled loss of mitochondrial membrane potential, reduced activity of SOD, and increased MDA and LDH. All these effects were inverted with overexpression of ZEB1.Conclusion: Altogether, the findings suggested that the up-regulation of miR-199a-3p significantly inhibited NSCLC growth in vivo, and reduced A549 cell proliferation and promoted mitochondrial-mediated apoptosis, through down-regulation of ZEB1. The findings supported ZEB1 down-expression with miR-199a-3p as a novel therapeutic target for NSCLC treatment.Keywords: lung cancer, miR-199a-3p, miR-199b-3p, ZEB1

Abstract 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.

Xiaoqing Gang, Mengmeng Yuan, Juxin Zhang Department of Gynecology, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou 450003, Henan, People’s Republic of ChinaCorrespondence: Juxin ZhangDepartment of Gynecology, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, No. 7 Weiwu Road, Jinshui District, Zhengzhou, Henan 450003, People’s Republic of ChinaTel +86-138 3818 4667Email zjuxin667@163.comObjective: Long non-coding RNAs (lncRNAs) have been identified as important players in tumorigenesis. LncRNA TMPO antisense RNA 1 (TMPO-AS1) has been shown to be involved in several tumors. However, the functional role and the underlying mechanism of TMPO-AS1 in regulating cervical cancer cell behavior remain unclear.Materials and Methods: Expression of TMPO-AS1, miR-143-3p, and ZEB1 were examined by qRT-PCR and Western blot. Cell proliferation, migration and invasion were evaluated using CCK-8 assay and Transwell migration and invasion assays, respectively. Luciferase reporter assay was performed to investigate the interaction miR-143-3p and TMPO-AS1 or ZEB1.Results: TMPO-AS1 was highly expressed in cervical cancer cells. Furthermore, TMPO-AS1 overexpression significantly promoted C-33A cell proliferation, migration, and invasion. In contrast, TMPO-AS1 silencing inhibited SiHa cell proliferation, migration, and invasion. Mechanistically, TMPO-AS1 acted as a sponge of miR-143-3p to elevate expression of zinc finger E-box binding homeobox 1 (ZEB1), a target of miR-143-3p, and thereby promoted C-33A cell proliferation, migration, and invasion. Further assays showed that TMPO-AS1 knockdown inhibited cervical cancer cell tumorigenesis in vivo.Conclusion: TMPO-AS1 promotes cervical cancer cell proliferation, migration, and invasion by regulating the miR-143-3p/ZEB1 axis.Keywords: cervical cancer, TMPO-AS1, miR-143-3p, ZEB1

Jingang Shen,1,* Xianbao Lv,1,* Lei Zhang2 1Department of Urology, Chengwu County People’s Hospital, Shandong 274200, People’s Republic of China; 2Department of Urology, Zoucheng People’s Hospital, Shandong 273500, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lei ZhangDepartment of Urology, Zoucheng People’s Hospital, No. 59, Qianquan Road, Zoucheng County, Jining, Shandong 273500, People’s Republic of ChinaTel +86-13506385177Email qifeng2001oy@163.comPurpose: GRHL2 played important roles in different cancers. In this study, we aimed to investigate the roles of GRHL2 in bladder cancer.Methods: The immunohistochemistry assay was performed to detect the expression of GRHL2 in bladder cancer tissues and adjacent noncancerous tissues and the expression levels of GRHL2 and zinc finger E-box binding homeobox (ZEB1) mRNA in tissues were determined by qRT-PCR. In addition, qRT-PCR and Western blotting were applied to detect the expression levels of GRHL2 and ZEB1 in bladder cancer cell lines (RT4, BIU-87, 5637, T24) and immortalized human bladder epithelial cell line (SV-HUC-1). The cell models with up-regulated and down-regulated expression of GRHL2 were constructed using bladder cancer cell lines T24 and 5637 to investigate the underlying roles of GRHL2 on the proliferation, migration, invasion and EMT process of bladder cancer cells. After that, cell proliferation was evaluated by CCK8 assay, cell cycle assay and colony formation assay. Transwell assay and wound healing assay were performed to determine the invasion and migration ability of the bladder cancer cells. The expressions of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, Vimentin, Slug and Snail) were assessed by Western blot analysis. Moreover, ZEB1 and GRHL2 were co-transfected into T24 and 5637 cells and their effects on EMT process and invasive capacity of cells were examined.Results: The expression of GRHL2 was down-regulated in bladder cancer tissues and human bladder cancer cell lines compared with the normal bladder tissues and immortalized human bladder epithelial cell line. Besides, down-regulation of GRHL2 improved the proliferation ability of bladder cancer cells and promoted the EMT process through up-regulation of ZEB1. The overexpression of ZEB1 partially reversed the inhibitory effect of GRHL2 on EMT.Conclusion: GRHL2 acts as an anti-oncogene to regulate bladder cancer cell proliferation and inhibit EMT by targeting ZEB1. This study may provide a theoretical basis for further research.Keywords: bladder cancer, GRHL2, EMT, ZEB1

Zhongping Ma,1 Guanghua Gu,1 Weikang Pan,1 Xiaoxiang Chen2 1Department of Obstetrics and Gynecology, Liyang Branch of Jiangsu Provincial People’s Hospital, Changzhou, People’s Republic of China; 2Department of Gynecologic Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing, People’s Republic of ChinaCorrespondence: Zhongping MaDepartment of Obstetrics and Gynecology, Liyang Branch of Jiangsu Provincial People’s Hospital, No. 70, Construction West Road, Liyang, Changzhou 213300, People’s Republic of ChinaTel +86 519 6809 1065Email gxuqub1972515kmz@yeah.netBackground: Cervical cancer (CC) is a common cancer with a poor prognosis due to the chemoresistance of CC cells to cisplatin. This study aimed to investigate the biological significance of lncRNA prostate cancer-associated transcript 6 (PCAT6) in the carcinogenesis of CC.Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to measure the abundance of PCAT6, miR-543 and zinc finger E-box binding protein 1 (ZEB1) in CC tissues and cells. The combination between miR-543 and lncRNA PCAT6 or ZEB1 was predicted by Starbase and was verified by dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay. Cell proliferation and chemoresistance to cisplatin were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis and metastasis were determined by flow cytometry, Western blot and transwell migration and invasion assays.Results: The abundance of ZEB1 protein was measured by Western blot assay. Murine xenograft model was established to confirm the function of lncRNA PCAT6 in vivo. The abundance of lncRNA PCAT6 was enhanced in CC tissues and cells compared with that in corresponding normal tissues and normal cervical epithelial cells Ect1/E6E7. MiR-543 was a target of PCAT6 and was negatively regulated by PCAT6. PCAT6 accelerated the proliferation, metastasis and the chemoresistance of CC cells to cisplatin while suppressed the apoptosis of CC cells. The overexpression of PCAT6 reversed the inhibitory effects of miR-543 accumulation on the proliferation, metastasis and chemoresistance of CC cells to cisplatin and the promoting impact on the apoptosis of CC cells. ZEB1 was a direct target of miR-543, and it functioned as the downstream gene of PCAT6/miR-543 to exert its oncogenic role in CC. PCAT6 promoted the growth of murine xenograft tumor through miR-543/ZEB1 axis in vivo.Conclusion: LncRNA PCAT6 facilitated the proliferation, metastasis and chemoresistance of CC cells to cisplatin while impeded the apoptosis of CC cells via PCAT6/miR-543/ZEB1 axis. PCAT6/miR-543/ZEB1 axis might be a promising target for CC therapy.Keywords: cervical cancer, lncRNA PCAT6, miR-543, ZEB1, chemoresistance

Abstract 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.

Thyroid cancer is now the most common endocrine malignancy and the effect of miR-429 in the development of thyroid cancer still need to be further investigated. The expression level of miR-429 was quantified by qPCR in both clinical samples and cultured cell lines. MTT, flow cytometry, migration analyses and Matrigel invasion assays were conducted to test the proliferation, apoptosis, migration and invasion of MiR-429 transfection in thyroid cancer cell lines. Luciferase activity assay and western blot were conducted to detect the direct effect of miR-429 on Zinc finger E-box-binding homeobox 1 (ZEB1) expression. In this study, it was found that miR-429 was frequently decreased in thyroid cancer tissues and cell lines. Transfection of miR-429 in thyroid cancer cell lines substantially suppressed cell proliferation, migration and invasion. Besides, miR-429 up-regulation would induce apoptosis in different cell lines. ZEB1 was identified as a direct target of miR-429 and miR-429 transfection could inhibit ZEB1 by direct binding to its 3’-untranslated region (3’-UTR). In conclusion, these data indicated that miR-429 could act as a tumour suppressor miRNA and contribute to the development and progression and metastasis of thyroid cancer.

BackgroundMany studies have reported the roles of the extracellular hypoxia microenvironment in the tumorigenesis and metastasis of multiple cancers. However, long noncoding RNAs (lncRNAs) that induce cancer oncogenicity and metastasis of pancreatic cancer (PC) under hypoxia conditions remain unclear.MethodsIn PC cells, the expression levels of lncRNAs in different conditions (normoxia or hypoxia) were compared through RNA sequencing (RNA-seq). The effects of the zinc finger E-box-binding homeobox 1 (ZEB1-AS1) antisense lncRNA on PC cells cultured in normoxia/hypoxia medium were measured through gain and loss-of-function experiments. Fluorescence in situ hybridization and luciferase reporter assays in addition to in vivo studies were utilized to explore the adaptive mechanisms of ZEB1-AS1 in the hypoxia-promoted proliferation, migration, and invasion ability of PC cells. Moreover, the level of ZEB1-AS1 and its associated targets or pathways were investigated in both PC and pancreatic normal tissues.ResultsRNA-seq revealed that ZEB1-AS1 was significantly upregulated in PC cells under hypoxia conditions. The ZEB1-AS1 expression level was closely associated with poor prognosis of PC patients. Knockdown of ZEB1-AS1 suppressed the proliferation, migration, and invasion of PC cells in vitro as well as PC xenograft tumor growth in vivo. In PC cells, RNAi-mediated reduction of ZEB1-AS1 inhibited zinc finger E-box-binding homeobox 1 (ZEB1), while ZEB1-AS1 overexpression rescued ZEB1 expression, indicating that ZEB1-AS1 promotes ZEB1 expression. Moreover, hypoxia-inducible factor-1α (HIF-1α)induced the expression of ZEB1-AS1 by binding to the ZEB1-AS1 promoter, which contains a putative hypoxia response element (HRE). Mechanistically, ZEB1-AS1 scaffolded the interaction among HIF-1α, ZEB1, and histone deacetylase 1 (HDAC1), leading to deacetylation-mediated stabilization of HIF-1α. We further revealed that ZEB1 induced the deacetylase capacity of HDAC1 to suppress the acetylation or degradation of HIF-1α, improving HIF-1α assembly. Thus, hypoxia-induced ZEB1-AS1 facilitated ZEB1 transcription and the stability of HIF-1α, which promoted the metastasis of PC cells. Clinically, dysregulated ZEB1 and HIF-1α expression was significantly correlated with histological grade, lymphatic metastasis, and distant metastasis in PC patients.ConclusionsOur results emphasized that the positive reciprocal loop of HIF-1α/ZEB1-AS1/ZEB1/HDAC1 contributes to hypoxia-promoted oncogenicity and PC metastasis, indicating that it might be a novel therapeutic target for PC.

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

Background: The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression., Methods: We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models., Results: Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture., Conclusions: Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)