OBJECTIVE

Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES.

RESULTS

The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor.

CONCLUSIONS

These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.

Human microRNA (miR)-141 is a member of the miR‑200 family, which has been reported to be downregulated in gastric cancer, and involved in the proliferation of gastric cancer cells. However, little is currently known regarding its role in the migration of gastric cancer. The present study investigated the function of miR‑141 in gastric cancer cell migration, and evaluated the contribution of zinc finger E‑box‑binding homeobox 1 and 2 (ZEB1/2) in miR‑141 mediated migration of gastric cancer cells. The expression levels of miR‑141 and its potential ZEB1/2 targets were examined by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. The migration of SGC‑7901 and HGC‑27 gastric cancer cells, which had been transfected with an miRNA precursor, was examined by cell migration and wound healing assays. A luciferase activity assay was used to validate whether ZEB1/2 was a direct target of miR‑141. The results demonstrated that overexpression of miR‑141 markedly inhibited the migration of gastric cancer cells in vitro. Forced overexpression of miR‑141 significantly reduced the luciferase activity of the 3'‑untranslated region of ZEB2 in gastric cancer cells. Furthermore, the mRNA and protein expression levels of ZEB2 were reduced in cells overexpressing miR‑141, whereas the protein expression levels of E‑cadherin were increased. In gastric tumor samples the expression levels of ZEB2 were inversely correlated with the expression of miR‑141. These results suggest that miR‑141 may be involved in the inhibition of gastric cancer cell migration, and that ZEB2 is a target gene of miR-141.

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC.

BACKGROUND

Epithelial-mesenchymal transition (EMT) plays a key role in the development of chronic obstructive pulmonary disease (COPD) and lung cancer.

OBJECTIVE

There are five major EMT regulatory genes (Snai1, Slug, Zeb1, Zeb2, and Twist1) involved in EMT. We hypothesized that germline variants in these genes may influence the development of both diseases.

METHODS

Seven genetic variants were genotyped in two two-stage case-control studies with 2,072 lung cancer cases and 2,077 control subjects, and 1,791 patients with COPD and 1,940 control subjects to show their associations with development of both diseases.

RESULTS

An exon variant c.353T>C(p.Val118Ala) of Snai1 harbored decreased risks of lung cancer (CT/CC vs. TT: odds ratio [OR], 0.76; 95% confidence interval [CI], 0.65-0.90) and COPD (CC vs. CT vs. TT: OR, 0.75; 95% CI, 0.63-0.89), and c.353T>C affected lung cancer risk indirectly through COPD (COPD accounted for 6.78% of effect that the variant had on lung cancer). Moreover, c.353T>C was correlated with lung cancer stages in smoking patients (P = 0.013), and those with the c.353C genotypes were less likely to have metastasis at diagnosis than those with the c.353TT genotype (OR, 0.60; 95% CI, 0.41-0.88). The c.353C allele encoding p.118Ala attenuated Snai1's ability to up-regulate mesenchymal biomarkers (i.e., fibronectin and vimentin) expression, and to promote EMT-like changes, including morphologic changes, cell migration, and invasion. However, these effects were not observed for the other variants.

CONCLUSIONS

The functional germline variant c.353T>C (p.Val118Ala) of Snai1 confers consistently decreased risks of lung cancer and COPD, and this variant affects lung cancer risk through a mediation effect of COPD.

Mowat-Wilson syndrome (MWS) is a genetic disease caused by heterozygous mutations or deletions of the ZEB2 gene and is characterized by distinctive facial features, epilepsy, moderate to severe intellectual disability, corpus callosum abnormalities and other congenital malformations. Epilepsy is considered a main manifestation of the syndrome, with a prevalence of about 70-75%. In order to delineate the electroclinical phenotype of epilepsy in MWS, we investigated epilepsy onset and evolution, including seizure types, EEG features, and response to anti-epileptic therapies in 22 patients with genetically confirmed MWS. Onset of seizures occurred at a median age of 14.5 months (range: 1-108 months). The main seizure types were focal and atypical absence seizures. In all patients the first seizure was a focal seizure, often precipitated by fever. The semiology was variable, including hypomotor, versive, or focal clonic manifestations; frequency ranged from daily to sporadic. Focal seizures were more frequent during drowsiness and sleep. In 13 patients, atypical absence seizures appeared later in the course of the disease, usually after the age of 4 years. Epilepsy was usually quite difficult to treat: seizure freedom was achieved in nine out of the 20 treated patients. At epilepsy onset, the EEGs were normal or showed only mild slowing of background activity. During follow-up, irregular, diffuse frontally dominant and occasionally asymmetric spike and waves discharges were seen in most patients. Sleep markedly activated these abnormalities, resulting in continuous or near-to-continuous spike and wave activity during slow wave sleep. Slowing of background activity and poverty of physiological sleep features were seen in most patients. Our data suggest that a distinct electroclinical phenotype, characterized by focal and atypical absence seizures, often preceded by febrile seizures, and age-dependent EEG changes, can be recognized in most patients with MWS.

Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease characterized by aberrant accumulation of fibroblast population and deposition of extra cellular matrix. Increasing evidence support that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is a critical process in the pathogenesis of IPF. Although delivery of bleomycin to induce acute lung injury is the most well-studied animal model of pulmonary fibrosis, there is considerable interest to pursue other models to understand the common and/or specific pathological mechanisms. In this study, we established a mouse model of pulmonary injury and progressive interstitial fibrosis via intraperitoneal injection of paraquat, a widely used herbicide known to cause pulmonary fibrosis in human. Using transcriptome sequencing and microarray analysis, we profiled expression of long non-coding RNAs (lncRNAs) and identified 513 up-regulated and 204 down-regulated lncRNAs in paraquat-induced fibrotic lung tissues. Gene ontology analysis revealed that the differentially expressed lncRNAs are implicated in cell differentiation, epithelium morphogenesis and wound healing, pathways closely associated with EMT. Furthermore, we identified the evolutionally conserved target genes of two up-regulated lncRNAs, uc.77 and 2700086A05Rik, as Zeb2 and Hoxa3, respectively, both of which are important modulators of EMT. Consistently, overexpression of uc.77 or 2700086A05Rik in human lung epithelial cells induced EMT as demonstrated by changes in gene and protein expression of various EMT markers and cell morphology. Collectively, our results uncovered a crucial role of lncRNA in the regulation of EMT during lung fibrosis and provide potential avenues for the discovery of novel molecular markers and therapeutic targets for IPF.

Circular RNAs (circRNAs) have been identified as crucial regulators of gene expression in human cancer biology. CircZFR is a novel identified circRNA and its effect in bladder cancer remains unclearly. In this study, we aimed to investigate the role of circZFR in the progression of bladder cancer. Firstly, we demonstrated that the expression of circZFR was higher in bladder cancer tissues and cells compared to adjacent non-tumor tissues and normal bladder epithelial cells. And higher circZFR levels were positively correlated with bladder cancer patients' pathological T stage, grade, lymphatic metastasis, recurrence, progression-free survival (PFS) and overall survival (OS). Functionally, knockdown of circZFR could significantly prohibit cell growth, migration and invasion, arrest cell cycle as well as promote apoptosis of bladder cancer cells in vitro study. Mechanistically, we observed that circZFR could directly bind to miR-377 as sponge to promote ZEB2 expression in bladder cancer cells. In addition, rescue assays demonstrated that restoration of ZEB2 significantly impaired the suppressive effects of circZFR silencing on bladder cancer cells growth, migration and invasion. Taken together, our results illuminated that circZFR could be a prognostic biomarker in bladder cancer and exerted oncogenic roles through regulating miR-377/ZEB2 axis in bladder cancer, which indicated that circZFR could be a potential therapeutic target for bladder cancer patients treatment.

Long non-coding RNA activated by transforming growth factor-beta (lnc-ATB) is abnormally expressed in multiple tumor types. The aim of this study was to investigate the expression of lnc-ATB and miR-141-3p, and to determine whether lnc-ATB can regulate epithelial-mesenchymal transition (EMT) by miR-141-3p in breast cancer. Here, we found that lnc-ATB was highly expressed whereas miR-141-3p was lowly expressed in breast cancer tissues and cells. Knockdown of lnc-ATB in two breast cancer cell lines (MDA-MB-231 and BT549) significantly increased miR-141-3p expression. Down-regulation of lnc-ATB resulted in a morphological change of breast cancer cells from spindle-like to round shape and in a remarkable inhibition of cell migration and invasion, which were reversed by miR-141-3p inhibitor. Furthermore, we demonstrated that lnc-ATB knockdown decreased ZEB1, ZEB2, N-cadherin, and vimentin expression, and promoted E-cadherin expression, while miR-141-3p inhibitor could reverse those effects. Moreover, we proved that miR-141-3p directly bound to the 3' untranslated region (UTR) of ZEB1 and ZEB2 and negatively regulated ZEB1 and ZEB2 expression. Taken together, our results show that knockdown of lnc-ATB significantly inhibits EMT process of breast cancer cells by increasing the expression of miR-141-3p, indicating that lnc-ATB might serve as a novel therapeutic target for breast cancer.

ZEB1 and ZEB2 have been recently related to cancer prognosis. We investigated their expression and its association with clinicopathological parameters and overall survival in invasive micropapillary carcinoma (IMPC), which is a metastasising neoplasm of the canine mammary gland. Immunohistochemical evaluation showed nuclear and cytoplasmic staining for ZEB2 and nuclear staining for ZEB1. 'In situ' areas presented higher positivity for cytoplasmic ZEB2 than invasive areas of IMPC did (p = 0.03). ZEB1 positivity was associated with a low histological grade (p = 0.01). A shorter overall survival rate was observed in IMPCs that were positive for cytoplasmic ZEB2 (p = 0.04). Antibodies specificity in canine species was confirmed by western blot. Our results indicated that cytoplasmic ZEB2 appears to be an important factor in the early stages of malignancy and predicts a poor overall survival rate for IMPC in this canine mammary cancer model. ZEB1 downregulation appears to be associated with the dedifferentiation process of IMPC.

The maintenance of myometrial quiescence and initiation of contractility, which lead to parturition at term and preterm, involve a shifting equilibrium between anti-inflammatory and proinflammatory signalling pathways. Progesterone (P4), acting through the progesterone receptor (PR), has an essential and multifaceted role in the maintenance of myometrial quiescence. This effect of P4-PR signalling is mediated, in part, by its anti-inflammatory actions and capacity to repress the expression of genes that encode proinflammatory cytokines, such as IL-1 and IL-6, and contraction-associated proteins, such as OXTR, GJA1 and PTGS2. By contrast, increased expression of genes that ultimately lead to parturition is mediated by enhanced inflammatory and estradiol-17β (E2) and estrogen receptor α signalling, which reduce PR function, thus further intensifying the inflammatory response. To obtain a more complete understanding of the molecular events that underlie the transition of the pregnant myometrium from a refractory to a contractile state, the roles of microRNAs, their targets, and their transcriptional and hormonal regulation have been investigated. This article reviews the actions of the miR-200 family and their P4-regulated targets-the transcription factors ZEB1, ZEB2 and STAT5B-in the pregnant myometrium, as well as the role of miR-199a-3p and miR-214 and their mutual target PTGS2. The central role of ZEB1 as the mediator of the opposing actions of P4 and E2 on myometrial contractility will be highlighted.

Epithelial ovarian cancer (EOC) is the fifth leading cause of female cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) are emerging as crucial regulators in various biological processes through diverse mechanisms. Recently, lncRNA Activated in RCC with Sunitinib Resistance (lncARSR) has been reported to be upregulated and involved in sunitinib resistance of renal cell carcinoma cells. However, the functional roles in EOC have not yet been explored. In the current study, we detected the expression levels of lncARSR in 76 paired EOC tissues and adjacent normal tissues, and observed that lncARSR expression was significantly increased in EOC tissues and correlated with FIGO stage, histological grade, lymph nodes metastasis and worse survival. Loss- and gain-of-function assays demonstrated that lncARSR promoted EOC cell proliferation and invasion. Further investigations showed that lncARSR interacted with HuR, upregulated β-catenin expression and then activated Wnt/β-catenin signaling pathway to regulate cell proliferation. Moreover, lncARSR increased ZEB1 and ZEB2 expression by competitively binding the miR-200 family to induce EMT and invasion. Our findings suggest that the lncARSR may provide a novel therapeutic strategy for EOC treatment.

BACKGROUND

The aim of this study was to explore the role of lncRNA zinc finger E-box binding homeobox 2 antisense RNA 1 (ZEB2-AS1), as a new tumor-associated lncRNA, in bladder cancer (BC) pathogenesis.

METHODS

BC tissues and tumor-adjacent normal bladder tissues were collected for detection of the expression profile of ZEB2-AS1 and miR-27b in BC. The endogenous expression of ZEB2-AS1 and miR-27b was modulated by the recombinant expression vector in vitro. The interaction between ZEB2-AS1 and miR-27b was identified by luciferase report gene assays and RNA immunoprecipitation (RIP) assays. The proliferation and apoptosis of BC cells was determined using CCK-8 assays and flow cytometric analysis.

RESULTS

The expression of ZEB2-AS1 was significantly increased in both BC tissues and BC cells (J82, 5637, T24); while miR-27b was down-regulated in BC tissues. More importantly, ZEB2-AS1 was significantly negative correlated with miR-27b expression in BC tissues (R2=0.1688, P<0.05). ZEB2-AS1 silencing inhibited BC cell proliferation and promoted apoptosis. Further studies confirmed that miR-27b was negatively regulated by ZEB2-AS1 in BC cells 5637 and T24, and the effects of ZEB2-AS1 on BC cells was mediated by miR-27b.

CONCLUSIONS

Our data provided strong evidence that ZEB2-AS1 promoted tumorigenesis and development of BC through down-regulating tumor-suppressive miR-27b.

Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the invasive phenotype and become migratory, which is required for cancer progression and metastasis. In the present study, the expression of EMT-associated biomarkers and their association with clinicopathological parameters in laryngeal squamous cell carcinoma (LSCC) was investigated. E-cadherin, N-cadherin, β-catenin and zinc finger E-box binding homeobox 2 (ZEB2) protein expression was evaluated with immunohistochemistry in a cohort of 76 patients with operable LSCC. The association between these transition markers, clinicopathological parameters and their prognostic impact in LSCC was analyzed. Immunohistochemical analysis revealed that EMT-associated proteins were differentially expressed between LSCC and adjacent non-neoplastic laryngeal tissue. Negative E-cadherin expression and positive N-cadherin, β-catenin and ZEB2 expression were associated with a later tumor (T) stage, decreasing tumor differentiation and a reduced overall survival (OS) time (OS: E-cadherin, P=0.016; N-cadherin, P=0.003; β-catenin, P=0.002; ZEB2, P=0.0003). E-cadherin/β-catenin co-expression was significantly associated with the majority of clinicopathological parameters assessed, including lymph node metastases, T stage and tumor cell differentiation (P=0.004, P=0.005, and P<0.001, respectively). Multivariate analysis indicated that T stage and the positive expression of β-catenin and ZEB2 were independent risk factors for OS in LSCC (P=0.014, P=0.025 and P=0.003, respectively). It was concluded that EMT mediates tumor progression, and reduces OS time in patients with LSCC. E-cadherin/β-catenin co-expression may be associated with clinicopathological parameters. T stage, and the positive co-expression of β-catenin and ZEB2 may be independent predictors of prognosis in LSCC.

Breast cancer is the leading cause of cancer death in women world wide which is closely related to metastasis. Recent studies argue that breast cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the molecular mechanisms underlying this transition are poorly understood. In this study, we found that increased expression of proline-rich protein 11 (PRR11) was associated with the progression of breast cancer and that PRR11 protein levels were significantly elevated in breast cancer. High PRR11 levels also predict shorter overall survival of breast cancer patients. Moreover, we found that the forced expression of PRR11 decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing PRR11 in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. PRR11 silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2).

Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1β stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.

Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.

OBJECTIVE

The homeobox B8 (HOXB8) functions as a sequence-specific transcription factor that is involved in development. Increased expression of this gene is associated with a wide variety of tumor; however, its function in gastric cancer has not been clarified. In the present study, the expression of HOXB8 in gastric cancer tissues and influence of HOXB8 on gastric cancer cellular were evaluated.

METHODS

The expression levels of HOXB8 mRNA in human gastric cancer tissues were analyzed through quantitative RT-PCR. To test the role of HOXB8 in gastric cancer metastasis, the cell transwell assay was performed. Microarray, ChIP-qPCR, and Western blot were used to explore the possible mechanism that HOXB8 promotes gastric cancer cells metastasis.

RESULTS

In this study, we found that HOXB8 showed higher expression in metastatic tissues than no-metastatic tissues. Overexpression of HOXB8 can promote gastric cancer cells migration and invasion, while silencing HOXB8 leads to the opposite results. Overexpression of HOXB8 also increases the rate of metastasis in NCI-N87 mice, while silencing HOXB8 has the opposite results. Furthermore, HOXB8 promotes epithelial-mesenchymal transformation of AGS cells. We also found that ZEB2 can interact with HOXB8 and may be a downstream factor of HOXB8 by using microarray. Knockdown of ZEB2 can inhibit HOXB8-induced migration and invasion capacity, as well as the epithelial-mesenchymal transformation in gastric cancer cells.

CONCLUSIONS

The results showed that HOXB8 plays an important role in the development and metastasis of gastric carcinoma.

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial differentiation is a protracted process in apes, involving a previously unrecognized transition state characterized by a change in cell shape. Furthermore, we show that human organoids are larger due to a delay in this transition, associated with differences in interkinetic nuclear migration and cell cycle length. Comparative RNA sequencing (RNA-seq) reveals differences in expression dynamics of cell morphogenesis factors, including ZEB2, a known epithelial-mesenchymal transition regulator. We show that ZEB2 promotes neuroepithelial transition, and its manipulation and downstream signaling leads to acquisition of nonhuman ape architecture in the human context and vice versa, establishing an important role for neuroepithelial cell shape in human brain expansion.

Keywords: ZEB2; brain; brain expansion; cell shape; chimpanzee; evolution; gorilla; neural stem cells; neuroepithelium; organoids.

Zinc finger E-box binding protein 1 (ZEB1) and ZEB2 induce epithelial-mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1-regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1-bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL-6 and IL-8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid-derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT.

MicroRNA-429 (miR-429) has been suggested to inhibit epithelial-mesenchymal transition (EMT), mainly due to targeting of ZEB1 and ZEB2, which are repressors of the cell to cell contact protein, E-cadherin. In this study, we indicated that regulation of miR-429 in cervical cancer cells modulates cell migration, elongation, as well as transforming growth factor β (TGF-β)-induced stress fiber formation through regulating the cytoskeleton reorganization which is likely independent of the zinc finger E-box binding homeobox (ZEB)/E-cadherin axis. ZEB1 and Crk-like adapter protein (CRKL), as novel targets of miR-429 and direct regulators of the actin cytoskeleton were identified. Remarkably, expression levels of ZEB1 and CRKL were inversely associated with the level of miR-429 in cervical cancer cell lines. In addition, individual knockdown and over-expression of these targeting genes phenocopied the roles of miR-429 over-expression and inhibition on cell elongation, migration, stress fiber formation, and invasion. Targeting of ZEB1 by miR-429 led to a decreased expression and transcriptional activity of CRB3, regulated by interference with the translocation of the CRB3. This finally led to decreasing of the expression of Crumbs 3 (CRB3), which is needed for the formation of stress fiber and contractility. Therefore, miR-429 affects cervical cancer by modulating some EMT-related processes. And in this study, evidences were provided to support a role for miR-429 as a novel target suppressing invasion and migration of human cervical cancer cells through modulation of its targeting genes ZEB1 and CRKL. Taken together, our data indicate that miR-429 plays a pivotal role in cervical cancer progression, which is a potential therapeutic target for patients.