LINC00472 suppressed by ZEB1 regulates the miR-23a-3p/FOXO3/BID axis to inhibit the progression of pancreatic cancer.

The tumour-suppressive role of LINC00472 has been extensively reported in various human cancers such as lung, colon and ovarian cancers, yet its function in pancreatic cancer remains unidentified. Here, the current research aimed to explore the role and regulatory axis mediated by LINC00472 in the progression of pancreatic cancer. RT-qPCR was adopted to determine LINC00472 expression in the harvested pancreatic cancer tissues and adjacent normal tissues. Loss-of-function and gain-of-function experiments were performed to examine the effects of LINC00472 on proliferation and apoptosis in vitro and tumorigenesis in vivo. Immunoblotting was performed to detect the expression of several proliferation and apoptosis-related proteins. Bioinformatic analysis, dual-luciferase reporter assay and RNA pull-down were conducted to profile the relationships between LINC00472 and miR-23a-3p, between miR-23a-3p and FOXO3 and between FOXO3 and BID. The LINC00472 expression was down-regulated by ZEB1 in the pancreatic cancer cells and tissues. LINC00472 could competitively bind to miR-23a-3p to enhance the expression of FOXO3, which consequently could promote the BID expression, thereby suppressing proliferation and promoting the apoptosis of pancreatic cancer cells. Meanwhile, the inhibitory role of LINC00472 in tumorigenesis was validated in vivo, and the LINC00472-mediated miR-23a-3p/FOXO3/BID axis was also demonstrated in the nude mouse tumour formation model. The study substantiated the antitumour activity of LINC00472 in pancreatic cancer and proposed a regulatory axis in which LINC00472 competitively binds to miR-23a-3p to enhance the FOXO3 expression and promote BID expression. Consequently, these findings provide theoretical basis for developing potential targets for the treatment of pancreatic cancer.

Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan-2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB-1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB-1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1-p21/ZEB-1 pathway, which provides new gene target therapy for PC.

Zeb1 promotes corneal neovascularization by regulation of vascular endothelial cell proliferation.

Angiogenesis is required for tissue repair; but abnormal angiogenesis or neovascularization (NV) causes diseases in the eye. The avascular status in the cornea is a prerequisite for corneal clarity and thought to be maintained by the equilibrium between proangiogenic and antiangiogenic factors that controls proliferation and migration of vascular endothelial cells (ECs) sprouting from the pericorneal plexus. VEGF is the most important intrinsic factor for angiogenesis; anti-VEGF therapies are available for treating ocular NV. However, the effectiveness of the therapies is limited because of VEGF-independent mechanism(s). We show that Zeb1 is an important factor promoting vascular EC proliferation and corneal NV; and a couple of small molecule inhibitors can evict Ctbp from the Zeb1-Ctbp complex, thereby reducing EC Zeb1 expression, proliferation, and corneal NV. We conclude that Zeb1-regulation of angiogenesis is independent of Vegf and that the ZEB1-CtBP inhibitors can be of potential therapeutic significance in treating corneal NV.

MiR-451a restrains the growth and metastatic phenotypes of papillary thyroid carcinoma cells via inhibiting ZEB1.

MicroRNAs (miRNAs) are known to be critical regulators in cancer progression. MiR-451a is reported to be involved in the progression of many different forms of cancers, including osteosarcoma, colorectal cancer, and breast carcinoma. In this study, we illuminated the possible roles of miR-451a in the development of papillary thyroid carcinoma (PTC) cells in vitro and in vivo. MiR-451a was markedly down-expressed in PTC sample compared with paratumor tissue. Upregulation of miR-451a repressed PTC cells proliferation, migration ability and inhibited the invasiveness of PTC cells in vitro. Additional, miR-451a suppressed PTC cells growth and the lung metastasis of PTC cells in vivo, whereas downregulation of miR-451a caused opposite outcomes. Importantly, miR-451a inversely modulated the expression of Zinc Finger E-Box Binding Homeobox 1 (ZEB1) by directly binding to the 3' untranslated region (UTR) of ZEB1 in PTC cells. The level of ZEB1 was negatively associated with miR-451a level in PTC tissues, and ZEB1 silencing mimicked the suppressive impacts of miR-451a on the proliferation, mobility, and invasive phenotypes of PTC cells. ZEB1 overexpression abrogated the inhibitory impacts of miR-451a on PTC cells. Together, this study revealed that miR-451a restrained the growth and metastatic phenotypes of PTC cells through targeting ZEB1.

ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins.

BACKGROUND: S100 proteins have been implicated in various aspects of cancer, including epithelial-mesenchymal transitions (EMT), invasion and metastasis, and also in inflammatory disorders. Here we examined the impact of individual members of this family on the invasion of pancreatic ductal adenocarcinoma (PDAC) cells, and their regulation by EMT and inflammation. METHODS: Invasion of PDAC cells was analysed in zebrafish embryo xenografts and in transwell invasion assays. Expression and regulation of S100 proteins was studied in vitro by immunoblotting, quantitative PCR and immunofluorescence, and in pancreatic lesions by immunohistochemistry. RESULTS: Whereas the expression of most S100 proteins is characteristic for epithelial PDAC cell lines, S100A4 and S100A6 are strongly expressed in mesenchymal cells and upregulated by ZEB1. S100A4/A6 and epithelial protein S100A14 respectively promote and represses cell invasion. IL-6/11-STAT3 pathway stimulates expression of most S100 proteins. ZEB1 synergises with IL-6/11-STAT3 to upregulate S100A4/A6, but nullifies the effect of inflammation on S100A14 expression. CONCLUSION: EMT/ZEB1 and IL-6/11-STAT3 signalling act independently and congregate to establish the expression pattern of S100 proteins, which drives invasion. Although ZEB1 regulates expression of S100 family members, these effects are masked by IL-6/11-STAT3 signalling, and S100 proteins cannot be considered as bona fide EMT markers in PDAC.

Regulation of Proliferation and Epithelial-to-Mesenchymal Transition (EMT) of Gastric Cancer by ZEB1 via Modulating Wnt5a and Related Mechanisms.

BACKGROUND As a member of the zinc-finger E-box binding protein (ZEB) family, ZEB1 can modulate onset and progression of various tumors, but its regulatory effect or mechanism in GC has not been defined. MATERIAL AND METHODS GC tumor tissues and adjacent tissues were collected from GC patients across different TNM stages. Real-time PCR was used to measure ZEB1 expression to analyze its correlation with pathological features of tumors. Cultured GC cell lines SGC-7901 and MGC-803 were randomly assigned into control group, scramble group, and ZEB1 siRNA group. Real-time PCR was employed to analyze ZEB1 expression, and MTT approach was used to measure cell proliferation. Cell apoptosis was evaluated by flow cytometry. Wound healing assay was used to detect its effect on cell migration. Expression of E-cadherin and Vimentin involved in epithelial-to-mesenchymal transition (EMT) was measured by Western blot analysis, along with Wnt5a proteins. RESULTS GC tissues had upregulation of ZEB1 (P<0.05 compared to adjacent tissues), whose expression level was correlated with differentiation grade, lymph node metastasis, and tumor pathological stage (P<0.05). Transfection of ZEB1 siRNA into SGC-7901 or MGC-803 cells can suppress ZEB1 expression, inhibit tumor cell proliferation, enhance apoptosis, and inhibit cell migration. Transfected GC cells had higher E-cadherin expression and decreased Vimentin expression or Wnt5a expression (P<0.05 compared to the control group). CONCLUSIONS ZEB1 expression is increased in GC tumor tissues and is associated with pathological features. The downregulation of ZEB1 can facilitate cell apoptosis via mediating Wnt5a, further suppressing GC cell proliferation and migration, and reducing EMT occurrence.

ZEB1 mediates doxorubicin (Dox) resistance and mesenchymal characteristics of hepatocarcinoma cells.

The acquired chemoresistance during long term chemotherapy is one of the most important factors to limit the application of Doxorubicin (Dox) on clinical treatment of hepatocellular carcinoma (HCC) patients. Our present study found that Dox resistant HCC (HCC/Dox) cells had greater capability of in vitro migration and invasion compared to their parental cells. HCC/Dox cells exhibited mesenchymal characteristics, which was evidenced by the up regulation of fibronectin, vimentin while down regulation of E-Cadherin. Zeb1, one powerful epithelial mesenchymal transition related transcription factor (EMT-TF), was markedly upregulated in HCC/Dox cells. Targeted inhibition of Zeb1 via siRNA can suppress the cell migration and re-sensitized cells to Dox treatment. The upregulation of Zeb1 in HCC/Dox cells was due to the increasing protein and mRNA stability of Zeb1. In HCC/Dox cells, the down regulation of SIAH1 mediated the upregulation of protein stability of Zeb1, while decreased levels of miR-3129-5p was responsible for the increasing mRNA stability of Zeb1. Collectively, our data suggested that SIAH1 and miR-3129-5p induced upregulation of Zeb1 mediated the Dox resistance of HCC cells. Targeted inhibition of Zeb1 might be helpful to overcome of Dox resistance of HCC.

The roles of ZEB1 in tumorigenic progression and epigenetic modifications.

Highly expressed Zinc-finger E-box binding protein 1 (ZEB1) is significantly associated with the malignancy of various cancers. Signal transduction and activation of ZEB1 play important roles in cancer transformation and epithelial-mesenchymal transition (EMT). Emerging evidence suggests that ZEB1 drives the induction of EMT with activation of stem cell traits, immune evasion and epigenetic reprogramming. As an ideal target for EMT research, ZEB1 has been extensively studied for decades. However, the link between ZEB1 and epigenetic regulation of EMT has only recently been discovered. ZEB1 facilitates the epigenetic silencing of E-cadherin by recruiting multiple chromatin enzymes of E-cadherin promoter, such as histone deacetylases (HDACs), DNA methyltransferase (DNMT) and ubiquitin ligase. Destruction of the connection between ZEB1 and these chromatin-modifying enzymes may represent an efficient for treating cancer. In this review, we outlined the biological function of ZEB1 in tumorigenic progression and epigenetic modifications and elucidate its transcriptional network, which is a suitable potential target for the design of novel anticancer drugs.

Silencing of the transcriptional factor ZEB1 alters the steroidogenic pathway, and increases the concentration of testosterone and DHT in DU145 cells.

Prostate cancer (PCa) is the second most common type of male malignancy worldwide. The transcription factor zinc finger E­box binding homeobox 1 (ZEB1) is associated with epithelial­mesenchymal transition and is also involved in regulation of androgen receptor (AR) expression, the main ligands of which are testosterone and dihydrotestosterone (DHT). These androgens are synthesized through the steroidogenic pathway within the prostate, and their synthesis is altered in PCa. The present study aimed to determine the ZEB1­induced alterations in androgen synthesis and AR expression in the DU145 PCa cell line. Reverse transcription­quantitative polymerase chain reaction, western blotting and immunocytochemistry were used to determine the mRNA and protein expression levels, and cellular localization of steroidogenic pathway enzymes in the DU145 cell line in response to ZEB1 silencing. Furthermore, the concentrations of testosterone and DHT were detected in cell culture medium using ELISA. ZEB1­silenced cells exhibited an increase in testosterone and DHT production, an increase in AR expression and an alteration in the steroidogenic pathway. In particular, steroidogenic acute regulatory protein and 5α­reductase 2 expression levels were decreased, whereas cytochrome P450 family 17 subfamily A member 1, 5α­reductase 1, aldo­keto reductase family 1 member D1 and aldo­keto reductase family 1 member C2 expression levels were increased. In conclusion, the present study provided novel information regarding the regulation of intratumoral androgen production in PCa, which is relevant for the progression of the disease to a castration­resistant form.

Inhibition of the Zeb family prevents murine palatogenesis through regulation of apoptosis and the cell cycle.

Mammalian palate separates the oral and nasal cavities for normal feeding, breathing and speech. The palatal shelves are a pair of maxillary prominences that consist of the neural crest-derived mesenchyme and surrounding epithelium. Palatogenesis is completed by the fusion of the midline epithelial seam (MES) after the medial edge epithelium (MEE) cells make contact between the palatal shelves. Various cellular and molecular events, such as apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT), are involved in palatogenesis. The Zeb family of transcription factors is an essential player during normal embryonic development. The distinct role of the Zeb family has not been thoroughly elucidated to date. In mouse palate, the Zeb family factors are expressed in the palatal mesenchyme until MEE contact. Interestingly, the expression of the Zeb family has also been observed in MES, which is already fused with the mesenchymal region. The regulatory roles of the Zeb family in palatogenesis have not been elucidated to date. The purpose of this study is to determine the Zeb family effects on the cellular events. To investigate the functions of the Zeb family, siRNA targeting Zeb family was used to treat in vitro organ culture for temporary inhibition of the Zeb family during palatogenesis. In the cultured palate containing siRNA, MES was clearly observed, and E-cadherin, an epithelial marker, was still expressed. Inhibition of the Zeb family results in the suppression of apoptosis, increased cell proliferation, and defective cell migration in the developing palate. Our data suggest that the Zeb family plays multiple roles in the stimulation and inhibition of apoptosis and cell proliferation and efficient mesenchymal cell migration during palatogenesis.