The activation of hepatic stellates cells (HSCs) is well believed to play a pivotal role in the development of liver fibrosis. MicroRNA-145 (miR-145) is known to suppress the progression of hepatocellular carcinoma, and is previously reported to be associated with Wnt/β-catenin pathway, but its role in the progression of hepatic fibrosis and activation of HSCs remains unknown and is warranted for investigation. In the present study, we found that the expression of miR-145 is significantly down-regulated in vivo in CCl4-induced mice liver fibrosis as well as in transforming growth factor-β1 (TGF-β1) induced HSC-T6 cell lines and human hepatic stellate cell line LX-2 in vitro. Furthermore, over-expression of miR-145 inhibited TGF-β1-induced the activation and proliferation of HSC-T6 cells in vitro. Mechanistically, we identified that zinc finger E-box-binding homeobox 2 (ZEB2), a key mediator of epithelial-to-mesenchymal transition, acted as a functional downstream target for miR-145. Interestingly, ZEB2 was shown to be involved in the TGF-β1-induced HSCs activation by regulating Wnt/β-catenin signaling pathway. Taken together, our results revealed the critical regulatory role of miR-145 in HSCs activation and implied miR-145 as a potential candidate for therapy of hepatic fibrosis by regulation of Wnt/β-catenin through targeting ZEB2.

Emerging evidence show that long noncoding RNAs (lncRNAs) play critical roles in tumor development. LincRNA-ROR (linc-ROR) is known to promote tumor progress in several human cancers, including hepatocellular carcinoma (HCC). Nevertheless, the roles of linc-ROR in HCC metastasis and its underlying mechanisms remain fully unclear. In the present study, we showed that linc-ROR was upregulated in HCC tissues and high linc-ROR expression level predicted poor prognosis. Functionally, linc-ROR significantly induced epithelial-mesenchymal transition (EMT), and increased in vitro invasion and in vivo metastasis of HCC cells. Mechanistically, linc-ROR acted as a sponge for miR-145 to de-repress the expression of target gene ZEB2, thereby inducing EMT and promoting HCC metastasis. Collectively, our research indicates the potential of linc-ROR as a vital therapeutic target for the treatment of aggressive and metastatic HCC.

Studies have shown that long noncoding RNA Zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) is involved in the progression of lung cancer, bladder cancer, and hepatocellular carcinoma. However, its role in the pathogenesis of gastric cancer remains unknown. The Wnt/β-catenin pathway contributes to the development of gastric cancer. ZEB2-AS1 expression was firstly detected in the gastric carcinoma tissue samples as well as in gastric cancer cells. Knockdown of ZEB2-AS1 was performed by ZEB2-AS1-shRNA, and the viability, migration, invasion, and apoptosis of gastric cancer cells were determined by CCK-8, scratch assay, transwell, and flow cytometry, respectively. Furthermore, levels of Ki-67, PCNA, VEGF, MMP9, epithelial-mesenchymal transition (EMT) markers (E-cadherin, Vimentin and ZEB2), cleaved caspase 3/8/9 and PARP, active β-catenin, c-Myc, cyclinD1, and AXIN2 were assayed by Western blot or real-time PCR. Additionally, the role and mechanism of ZEB2-AS1 were confirmed in a xenograft nude mouse model. We found ZEB2-AS1 expression was increased in gastric carcinoma samples, and it was correlated with tumor progression. Also, its expression was elevated in gastric cancer cells. Knockdown of ZEB2-AS1 reduced the proliferation, migration, invasion, and EMT, but increased the apoptosis of gastric carcinoma cells. Furthermore, ZEB2-AS1 downregulation remarkably suppressed the expression of Ki-67, PCNA, VEGF and MMP9, and the activation of Wnt/β-catenin signaling, whereas elevated the levels of cleaved caspase 3/8/9 and PARP in gastric cancer cells. And ZEB2 overexpression reversed the effects of ZEB2-AS1 downregulation on the proliferation, EMT and inactivation of Wnt/β-catenin signaling. Additionally, ZEB2-AS1 knockdown inhibited tumor growth, Ki-67 staining, and the expression of VEGF, MMP9, active β-catenin, c-Myc, cyclinD1, and AXIN2 in mice. In conclusion, ZEB2-AS1 promotes the tumorigenesis of gastric carcinoma that is related to the upregulation of ZEB2 and the activation of the Wnt/β-catenin pathway.

Background: Circular RNAs (circRNAs) have been reported as the competing endogenous RNAs (ceRNAs) to sponge microRNAs (miRNAs) implicating in the initiation and progression of breast cancer. However, the functions of circRNAs in breast cancer have not been completely clarified. In the present study, we aimed to identify differentially expressed circRNAs in breast cancer tumor tissues, and their roles and downstream targets were investigated in the progression of breast cancer. Methods: High-throughput circRNA sequencing was performed to detect the differentially expressed circRNAs. The CCK-8 and flow cytometry were performed to measure the cell viability and apoptosis in breast cancer cells. Gene and protein expression were assayed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Results: hsa_circ_0004771 and Zinc finger E-box binding homeobox 2 (ZEB2) expression levels were up-regulated and positively correlated in breast cancer tumor tissues. In addition, the expression levels of miR-653 were reduced in breast cancer tumor tissues. We also found that hsa_circ_0004771 functioned as a sponge of miR-653 to inhibit its expression. miR-653 as a post-transcriptional regulator down-regulated the expression of ZEB2 by binding to its 3'-UTR. Interestingly, a significant inverse correlation was observed between miR-653 and hsa_circ_0004771 or ZEB2 expression in breast cancer tumor tissues. Knockdown of hsa_circ_0004771 and ZEB2 served as equally authentic of miR-653 mimics to induce growth inhibition and apoptosis in breast cancer cells. Conclusion: Hsa_circ_0004771/miR-653/ZEB2 regulatory feedback revealed a new molecular mechanism in the pathogenesis of breast cancer, which might provide novel therapeutic targets for the treatment of breast cancer.

OBJECTIVE

Epithelial-mesenchymal transition (EMT), characterized by the decrease of E-cadherin (E-Cad) and increase in vimentin and alpha-smooth muscle actin (α-SMA), was demonstrated to participate in inflammatory bowel disease-related fibrosis. miR-200b plays an anti-fibrosis role in inhibiting EMT by targeting ZEB1 and ZEB2. But the stability of exogenous miR-200b in blood limits its application. Microvesicles (MVs), which can transfer miRNAs among cells and prevent them from degradation, may provide an excellent transport system for the delivery of miR-200b in the treatment of fibrosis.

METHODS

Bone marrow mesenchymal stem cells (BMSCs) were transfected with lentivirus to overexpress miR-200b. The MVs packaged with miRNA-200b were harvested for the anti-fibrotic treatment using in vitro (transforming growth factor beta 1-mediated EMT in intestinal epithelial cells: IEC-6) and in vivo (TNBS-induced intestinal fibrosis in rats) models. The pathological morphology was observed, and the fibrosis related proteins, such as E-Cad, vimentin, α-SMA, ZEB1, and ZEB2, were detected.

RESULTS

MiR-200b-MVs would significantly reverse the morphology in TGF-β1-treated IEC-6 cells and improve the TNBS-induced colon fibrosis histologically. The treatment of miR-200b-MVs increased miR-200b levels both in the IEC-6 cells and colon, resulting in a significant prevention EMT and alleviation of fibrosis. The expression of E-Cad was increased, and the expressions of vimentin and α-SMA were decreased. ZBE1 and ZEB2, the targets of miR-200b, were also decreased.

CONCLUSIONS

miR-200b could be transferred from genetically modified BMSCs to the target cells or tissue by MVs. The mechanisms of miR-200b-MVs in inhibiting colonic fibrosis were related to suppressing the development of EMT by targeting ZEB1and ZEB2.

BACKGROUND

Understanding the molecular mechanisms is important in development and therapy of endometrioid endometrial adenocarcinoma.

OBJECTIVE

To identify key genes in endometrioid endometrial adenocarcinoma.

METHODS

The data of mRNA, miRNA and DNA methylation were downloaded from The Cancer Genome Atlas (TCGA) database and differential analysis was performed. Then, bioinformatic analysis was used to explore the regulatory mechanisms of miRNA and DNA methylation on gene expression. The regulatory network between differentially expressed miRNAs and target genes was established. Finally, the quantitative RT-PCR was applied to validate the bioinformatics results.

RESULTS

We obtained biological omics data of 381 patients with endometrioid endometrial adenocarcinoma from TCGA data portal. After data processing, up to 2068 DEGs and 69 differentially expressed miRNAs were identified. Prediction and correlation analysis revealed that 175 DEGs that were not only the target genes but also negatively correlated with the screened differentially expressed miRNAs. After the integrated analysis of differentially methylated CpG islands and DEGs, 16 related genes were obtained. The quantitative RT-PCR results were roughly consistent with the bioinformatics analysis.

CONCLUSIONS

The altered DEGs (ZEB1, ZEB2, TIMP2, TCF4, CYP1B1, PITX1, PITX2, ZNF154 and TSPYL5) may be involved in tumor differentiation of endometrioid endometrial adenocarcinoma and could be used as potential therapeutic targets for the disease.

The transcription factor hepatocyte nuclear factor-1β (HNF-1β) regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1β produce kidney cysts, and previous studies have shown that HNF-1β regulates the transcription of cystic disease genes, including Pkd2 and Pkhd1. Here, we combined chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) with microarray analysis to identify microRNAs (miRNAs) that are directly regulated by HNF-1β in renal epithelial cells. These studies identified members of the epithelial-specific miR-200 family (miR-200b/200a/429) as novel transcriptional targets of HNF-1β. HNF-1β binds to two evolutionarily conserved sites located 28 kb upstream to miR-200b. Luciferase reporter assays showed that the HNF-1β binding sites were located within a promoter that was active in renal epithelial cells. Mutations of the HNF-1β binding sites abolished promoter activity. RT-PCR analysis revealed that a long noncoding RNA (lncRNA) is transcribed from the promoter and encodes the miR-200 cluster. Inhibition of the lncRNA with siRNAs decreased the levels of miR-200 but did not affect expression of the Ttll10 host gene. The expression of the lncRNA and miR-200 was decreased in kidneys from HNF-1β knock-out mice and renal epithelial cells expressing dominant-negative mutant HNF-1β. The expression of miR-200 targets, Zeb2 and Pkd1, was increased in HNF-1β knock-out kidneys and in cells expressing mutant HNF-1β. Overexpression of miR-200 decreased the expression of Zeb2 and Pkd1 in HNF-1β mutant cells. These studies reveal a novel pathway whereby HNF-1β directly contributes to the control of miRNAs that are involved in epithelial-mesenchymal transition and cystic kidney disease.

The epithelial-to-mesenchymal transition (EMT) program is critical for epithelial cell cancer progression and fibrotic diseases. FOXO1 influences a broad range of physiological and pathological processes. However, the mechanism by which FOXO1 inhibits EMT is not fully understood. In this study, we demonstrated that FOXO1 overexpression inhibited cell motility and invasiveness in vitro and inhibited lung metastasis in vivo. In addition, we found that FOXO1 couldreverse the EMT program. FOXO1 silencing by siRNA in hepatocellular carcinoma (HCC) cell lines enhanced the expression of mesenchymal markers and decreased the expression of the epithelial markers. Consistent with these findings, FOXO1 overexpression exerted opposite effects. Furthermore, we found that FOXO1 levels were inversely correlated with the levels of EMT inducers, including Snail, Slug, ZEB1, ZEB2 and Twist1 in HCC cells. Co-immunoprecipitation and immunohistochemistry assays revealed that an interaction between FOXO1 and ZEB2. A dual-luciferase reporter assay and a ChIP assay further demonstrated that FOXO1 binds to the ZEB2 promoter. Together, these findings suggest that FOXO1 overexpression or ZEB2 inhibition might be potential therapeutic strategies for treating HCC.

OBJECTIVE

What is the role of miR-429 in murine embryo implantation?

CONCLUSIONS

miR-429 functions as a suppressor of epithelial-mesenchymal transition (EMT) during the process of embryo implantation by reverse regulation of Pcdh8.

BACKGROUND

MicroRNAs (miRNAs) may serve as promising regulators of embryo implantation. miR-429 was recently found to be down-regulated during embryo implantation period in a microarray analysis.

METHODS

The expression profile of miR-429 was clarified in a series of models, and the target gene was confirmed. The in vivo and in vitro effect of miR-429 on embryo implantation was examined.

METHODS

Pregnancy was produced by natural mating between female C57BL6/J mice and male mice, and a series of models, including pseudopregnancy, delayed implantation and artificial decidualization, were established. The expression profile of miR-429 during the embryo implantation period was clarified in these models. Candidate target genes of miR-429 were predicted by bioinformatic analysis and tested by luciferase activity assay. The in vivo effects of miR-429 on embryo implantation were also examined. The in vitro effects of miR-429 on EMT were studied by examining migratory and invasive capacities by transwell assay and expression profiles of cadherin family members by western blotting and qRT-PCR.

RESULTS

The expression profile of miR-429 in animal models suggested its down-regulation should be dependent on the presence and status of blastocysts and on endometrial decidualization. The luciferase activity assay showed that Pcdh8, a member of cadherin gene family, was the target gene of miR-429, and miR-429 suppressed the expression of Pcdh8 mRNA and protein. Gain-of-function of miR-429 in vivo resulted in a significant reduction of the number of implantation sites, but had little effect on fertilization. Up-regulation of miR-429 in vitro led to suppression of mesenchymal marker genes Vim, Cdh2, Zeb1 and Zeb2, and activation of epithelial marker gene Cdh1, resulting in suppression of the migratory and invasive capacities of cells. miR-429 also partially abrogated TGF-beta-induced EMT. The dysregulated expression profiles of EMT markers during embryo implantation period could be partially reversed by gain-of-function of miR-429 in vivo.

CONCLUSIONS

The association of miR-429 with other members of the miR-200 family in embryo implantation remains to be determined. The relationship between miR-429 and the cadherin family needs more intensive description and the detailed mechanism of miR-429 in regulating the cadherin family needs to be elucidated.

CONCLUSIONS

Our findings indicate that miR-429 plays a major role in embryo implantation as a suppressor of EMT by targeting Pcdh8. This information could contribute to a better understanding of the mechanisms involved in the miRNA-mediated regulation of embryo implantation, and subsequently improve treatments for infertility. The findings are consistent with that from previous research of the other members in miR-200 family in embryo implantation and in the EMT.

BACKGROUND

This study was supported by the Natural Science Foundation of China (Grant number: 81170592), and Special Fund from National Excellent Doctoral Dissertation (Grant number: 201079). There was no conflict of interest.

Increasing evidence shows that micro RNAs (miRNAs) play a critical role in tumor development. However, the role of miRNAs in non-small cell lung cancer (NSCLC) metastasis remains largely unknown. Here, we found that miR-124 expression was significantly impaired in NSCLC tissues and associated with its metastasis. In vitro and in vivo experiments indicate that restoring miR-124 expression in NSCLC cells had a marked effect on reducing cell migration, invasion and metastasis. Mechanistic analyses show that Smad4, a cobinding protein in transforming growth factor-β (TGF-β) pathway, was identified as a new target gene of miR-124. Restoring Smad4 expression in miR-124-infected cells could partially rescue miR-124-induced abolition of cell migration and invasion. Notably, upon TGF-β stimulation, phosphorylation of Smad2/3 was modulated by alteration of miR-124 or Smad4 expression, followed by inducing some special transcription of downstream genes including Snail, Slug and ZEB2, all of which may trigger epithelial-mesenchymal transition and be associated with NSCLC metastasis. Moreover, activation of TGF-β pathway may enhance expression of DNMT3a, leading to hypermethylation on miR-124 promoter. Therefore, heavily loss of miR-124 expression further enhances Smad4 level by this feedback loop. Taken together, our data show for the first time that the feedback loop between miR-124 and TGF-β pathway may play a significant role in NSCLC metastasis. Targeting the loop may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for NSCLC.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1⁺ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8⁺ tissue-resident memory T (T_RM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8⁺ T_RM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8⁺ T_RM cells in IBD.

This study was specifically designed to confirm the hypothesis that microRNA-200c (miR-200c) affects the development of cisplatin (DDP) resistance in human gastric cancer cells by targeting zinc finger E-box binding homeobox 2 (ZEB2). A total of 50 gastric cancer tissues and their corresponding normal adjacent tissue samples were collected. Then, the expression levels of miR-200c and ZEB2 in both gastric cancer specimens and cells were detected using the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical methods. A dual‑luciferase reporter gene assay was conducted to evaluate the effect of miR-200c on the 3'-untranslated region (3'UTR) luciferase activity of ZEB2. SGC7901/DDP cells were transfected with miR-200c mimics and ZEB2 siRNA, respectively. Subsequently, changes in cellular proliferation and apoptosis were detected through the methyl thiazolyl tetrazolium assay and flow cytometric analysis, respectively. We also carried out a western blot analysis assay in order to detect the expression of apoptosis-related genes and ZEB2. miR-200c was significantly downregulated and ZEB2 was significantly upregulated in both gastric cancer tissues and SGC7901/DDP cells when compared with those in normal tissues and SGC7901 cells (P<0.01). The dual luciferase reporter gene assay showed that miR-200c could specifically bind with the 3'UTR of ZEB2 and significantly suppress the luciferase activity by 42% (P<0.01). Upregulation of miR-200c or downregulation of ZEB2 enhanced the sensitivity of SGC7901/DDP cells to DDP. miR‑200c was significantly downregulated in both gastric cancer tissues and cells, while the expression of ZEB2 exhibited the opposite trend. Our study further demonstrated that miR-200c could enhance the sensitivity of SGC7901/DDP cells to DDP through targeted regulation of ZEB2 expression in gastric cancer tissues.

BACKGROUND

Recent studies have verified that long noncoding RNAs (lncRNAs) involved in many biological functions and play crucial roles in human cancers progression, the study aimed to detect the association between long non-coding RNA HOXA11-AS and epithelial-mesenchymal transition (EMT) process in non-small cell lung cancer (NSCLC).

METHODS

The lncRNA HOXA11-AS expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays in 78 paired of tumor tissue and adjacent normal tissue samples in NSCLC patients. Kaplan-Meier survival curves and log-rank test was used to examine the association between lncRNA HOXA11-AS expression and the over survival time in NSCLC patients. Transwell invasion assay was performed to detect the cell invasion ability. QRT-PCR and western-blot analysis detected the mRNA and protein expression of EMT related transcription factors ZEB1/ZEB2, Snail1/2 and EMT marker E-cadherin and N-cadherin in NSCLC cells. RIP and Chromatin immunoprecipitation assays were performed to analyze the association between lncRNA HOXA11-AS and miR-200b expression in NSCLC cells.

RESULTS

The lncRNA HOXA11-AS expression levels were significantly higher in NSCLC tissues compared with adjacent normal tissues and higher HOXA11-AS expression levels had a poor prognosis in NSCLC patients. Furthermore, knockdown of lncRNA HOXA11-AS in A549 and H1299 cells dramatically inhibited cell invasive abilities. Besides, the transcription levels and protein levels of EMT related transcription factors ZEB1/ZEB2, Snail1/2, and EMT maker N-cadherin were down-regulated after lncRNA HOXA11-AS was knocked down, but the mRNA and protein expression levels of EMT maker E-cadherin was increasing in A549 and H1299 cells. The mechanistic findings showed demonstrated that HOXA11-AS interacted with EZH2 and DNMT1 and recruited them to the miR-200b promoter regions to repress miR-200b expression in NSCLC cells, which promoted cell EMT in NSCLC.

CONCLUSIONS

Our results showed that up-regulation of lncRNA HOXA11-AS predicted a poor prognosis and lncRNA HOXA11-AS promoted cell epithelial-mesenchymal transition (EMT) by inhibiting miR-200b expression in NSCLC.

The overall goal of the present study was to find and validate unidentified miRNAs that regulate epithelial-mesenchymal transition (EMT) and proliferation in ovarian cancer. Furthermore, we demonstrate that the high expression of miR-153 in human epithelial ovarian cancer (EOC) is associated with better survival. The mean expression level of miR-153 in ovarian cancer was significantly lower than in the adjacent carcinoma tissue. In the present study, we report that miR-153 are negative regulators of SET7 and ZEB2, miR-153 regulates SET7/ZEB2 expression and promotes SET7/ZEB2 mRNA degradation. Further, confirmed by reporter assays, SET7/ZEB2 are downstream targets of miR-153 directly bound to the 3' untranslated region (3'-UTR). Clone formation and wound-healing assay as well as Transwell assay proved that silencing of SET7 or ZEB2 partially abolished the enhancement of cell proliferation and invasion induced by downregulated miR-153. SET7 and ZEB2 are negatively correlated with miR-153 expression in human ovarian cancer and indicated a worse survival. Considering the role of SET7 and ZEB2 in EOC, it is important to clarify how the expression of SET7 and ZEB2 are regulated. Based on our results miR-153 inhibits proliferation and suppresses EMT and the invasive potential of ovarian cancer cells through downregulation of SET7 and ZEB2, supporting the pursuit of miR-153 as a potential target for ovarian cancer intervention.

The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar epithelial (MLE-12) cell line or human renal proximal tubule (HK2) cell line transiently or stably lacking the expression of SIK1 (using SIK1 siRNAs or shRNAs), or with its expression abrogated (sik1(+/+) vs. sik1(-/-) mice), indicate that suppression of SIK1 (∼40%) increases the expression of the transcriptional repressors Snail2 (∼12-fold), Zeb1 (∼100%), Zeb2 (∼50%), and TWIST (∼20-fold) by activating cAMP-response element binding protein. The lack of SIK1 and activation of transcriptional repressors decreases the availability of E-cadherin (mRNA and protein expression by ∼100 and 80%, respectively) and the stability of intercellular junctions in epithelia (decreases in transepithelial resistance). Furthermore, LKB1-mediated increases in E-cadherin expression are impaired in cells where SIK1 has been disabled. We conclude that SIK1 is a key regulator of E-cadherin expression, and thereby contributes to the stability of intercellular junctions.

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process by which a fully differentiated epithelial cell acquires mesenchymal traits, and therefore, mesenchymal abilities such as motility and invasiveness. It is a pivotal physiological process involved in embryogenesis (Type 1 EMT) and in wound healing and tissue remodeling (Type 2 EMT), which, some authors claim, but there are still some controversies, has also been co-opted by tumor cells to increase their malignant potential (Type 3 EMT). Many biomarkers of Type 3 EMT have been characterized and classified into functional categories (i.e., extracellular proteins, cell surface molecules, cytoskeletal markers, transcriptional factors, and, recently, micro RNAs). The extra and intracellular signals that lead to EMT are only starting to be understood, but there is a consensus that Ras and TGF-beta signaling must converge with NF-κB in order to achieve a full EMT. The most classical experimental model is the induction of EMT by TGF-beta in cultures of epithelial cells. Other pathways involving GSK3b, and Wnt/beta-catenin, are also implicated. Ultimately, every EMT-inducing pathway will activate any of the E-cadherin transcriptional repressors (ZEB1, ZEB2, Twist, Snail or Slug). Although in the pre-clinical setting, EMT has also been related to an accelerated tumor progression and to an increased resistance to conventional chemotherapy. In this sense, several groups are beginning to use EMT as a predictive marker of response to treatment. Finally, two chemicals targeting TGF-beta are in clinical trials and many laboratories have initiated studies to use other EMT-related molecules as a therapeutic target for the cancer patient with some modest, but encouraging results.

Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ~70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

Resistance to BRAF inhibitors (BRAFi) is one of the major challenges for targeted therapies for BRAF-mutant melanomas. However, little is known about the role of microRNAs in conferring BRAFi resistance. Herein, we demonstrate that miR-200c expression is significantly reduced whereas miR-200c target genes including Bmi1, Zeb2, Tubb3, ABCG5, and MDR1 are significantly increased in melanomas that acquired BRAFi resistance compared to pretreatment tumor biopsies. Similar changes were observed in BRAFi-resistant melanoma cell lines. Overexpression of miR-200c or knock-down of Bmi1 in resistant melanoma cells restores their sensitivities to BRAFi, leading to deactivation of the PI3K/AKT and MAPK signaling cascades, and acquisition of epithelial-mesenchymal transition-like phenotypes, including upregulation of E-cadherin, downregulation of N-cadherin, and ABCG5 and MDR1 expression. Conversely, knock-down of miR-200c or overexpression of Bmi1 in BRAFi-sensitive melanoma cells activates the PI3K/AKT and MAPK pathways, upregulates N-cadherin, ABCG5, and MDR1 expression, and downregulates E-cadherin expression, leading to BRAFi resistance. Together, our data identify miR-200c as a critical signaling node in BRAFi-resistant melanomas impacting the MAPK and PI3K/AKT pathways, suggesting miR-200c as a potential therapeutic target for overcoming acquired BRAFi resistance.

Polled and Multisystemic Syndrome (PMS) is a novel developmental disorder occurring in the progeny of a single bull. Its clinical spectrum includes polledness (complete agenesis of horns), facial dysmorphism, growth delay, chronic diarrhea, premature ovarian failure, and variable neurological and cardiac anomalies. PMS is also characterized by a deviation of the sex-ratio, suggesting male lethality during pregnancy. Using Mendelian error mapping and whole-genome sequencing, we identified a 3.7 Mb deletion on the paternal bovine chromosome 2 encompassing ARHGAP15, GTDC1 and ZEB2 genes. We then produced control and affected 90-day old fetuses to characterize this syndrome by histological and expression analyses. Compared to wild type individuals, affected animals showed a decreased expression of the three deleted genes. Based on a comparison with human Mowat-Wilson syndrome, we suggest that deletion of ZEB2, is responsible for most of the effects of the mutation. Finally sperm-FISH, embryo genotyping and analysis of reproduction records confirmed somatic mosaicism in the founder bull and male-specific lethality during the first third of gestation. In conclusion, we identified a novel locus involved in bovid horn ontogenesis and suggest that epithelial-to-mesenchymal transition plays a critical role in horn bud differentiation. We also provide new insights into the pathogenicity of ZEB2 loss of heterozygosity in bovine and humans and describe the first case of male-specific lethality associated with an autosomal locus in a non-murine mammalian species. This result sets PMS as a unique model to study sex-specific gene expression/regulation.

Zinc finger E-box-binding homeobox 2 (ZEB2) was closely related to the oncogenesis, development and response to chemotherapy of cancer. However, its biological functions in small cell lung cancer (SCLC) remain unknown. The aim of this study is to investigate the roles of ZEB2 in chemoresistance of SCLC and its possible molecular mechanism. Expression of ZEB2 was examined in sixty-eight cases of SCLC tissues by immunohistochemistry. Knockdown of ZEB2 was carried out in SCLC multidrug resistant cells (H69AR) to assess its influence on chemoresistance. The results showed that ZEB2 was expressed in 23.5% (16/68) of SCLC. Overexpression of ZEB2 was associated with the poor pathologic stage of SCLC (P < 0.001 by the Fisher's Exact Test) and the shorter survival time (by the Kaplan-Meier method). Inhibition of ZEB2 expression using small interfering RNA in H69AR cells sensitized cancer cells to chemotherapeutic drugs through increasing drug-induced cell apoptosis accompanied with S phase arrest. In silico analysis demonstrated that there are complementary binding sites between miR-200b and ZEB2 3'-UTR, and identified miR-200b as a potential regulator of ZEB2. We found that miR-200b was down-regulated in the resistant cells and enforced expression of miR-200b by miRNA mimics increased cell sensitivity. Overexpression of miR-200b led to the downregulation of ZEB2 at protein level. Luciferase reporter gene assay showed that 3'UTR ZEB2 activity was regulated by miR-200b. Our results suggest that ZEB2 modulates drug resistance and is regulated by miR-200b. All findings provide insight into the ZEB2 signaling mechanism and ZEB2 may be a potentially novel target for multi-drug resistance in SCLC.

BACKGROUND

Chronic airway inflammatory disorders, such as asthma, are characterized by airway inflammation and remodeling. Chronic inflammation and damage to the airway epithelium cause airway remodeling, which is associated with improper epithelial repair, and is characterized by elevated expression of transforming growth factor-β (TGF-β). Epithelial-mesenchymal transition (EMT) is an important mechanism during embryonic development and tissue remodeling whereby epithelial cells gain the capacity to increase motility by down-regulation of epithelial markers and up-regulation of mesenchymal markers. TGF-β is a central inducer of EMT, and TGF-β-induced EMT is enhanced by pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β. We investigated whether the pro-inflammatory cytokine TWEAK (TNF-like weak inducer of apoptosis) enhanced TGF-β1-induced EMT in the human bronchial epithelial cell line BEAS-2B.

METHODS

Quantitative RT-PCR and western blotting were used to define alterations in epithelial and mesenchymal marker expression in BEAS-2B cells. The cells were assessed for 48 h after stimulation with TGF-β1 alone or in combination with TWEAK.

RESULTS

TGF-β1 induced spindle-like morphology and loss of cell contact, and reduced the expression of epithelial marker E-cadherin and increased the expression of mesenchymal markers N-cadherin and vimentin. Our data, for the first time, show that TWEAK reduced the expression of E-cadherin, and that co-treatment with TGF-β1 and TWEAK enhanced the TGF-β1-induced features of EMT. Moreover, hyaluronan synthase 2 expression was up-regulated by a combination with TGF-β1 and TWEAK, but not TNF-α. We also demonstrated that the Smad, p38 MAPK, and NF-κB signaling pathways, and the transcriptional repressor ZEB2 might mediate N-cadherin up-regulation by TGF-β1 in combination with TWEAK.

CONCLUSIONS

These findings suggest that the pro-inflammatory cytokine TWEAK and TGF-β1 have synergistic effects in EMT and may contribute to chronic airway changes and remodeling.

Diffuse invasion of the surrounding brain parenchyma is a major obstacle in the treatment of gliomas with various therapeutics, including anti-angiogenic agents. Here we identify the epi-/genetic and microenvironmental downregulation of ephrinB2 as a crucial step that promotes tumour invasion by abrogation of repulsive signals. We demonstrate that ephrinB2 is downregulated in human gliomas as a consequence of promoter hypermethylation and gene deletion. Consistently, genetic deletion of ephrinB2 in a murine high-grade glioma model increases invasion. Importantly, ephrinB2 gene silencing is complemented by a hypoxia-induced transcriptional repression. Mechanistically, hypoxia-inducible factor (HIF)-1α induces the EMT repressor ZEB2, which directly downregulates ephrinB2 through promoter binding to enhance tumour invasiveness. This mechanism is activated following anti-angiogenic treatment of gliomas and is efficiently blocked by disrupting ZEB2 activity. Taken together, our results identify ZEB2 as an attractive therapeutic target to inhibit tumour invasion and counteract tumour resistance mechanisms induced by anti-angiogenic treatment strategies.

Tumor metastasis, a complex process involving the spread of malignant tumor cells from a primary tumor site to a distant organ, is a major cause of failure of cancer chemotherapy. Epithelial-mesenchymal transition (EMT) is a critical step for the initiation of cancer metastasis. The processes of EMT and metastasis are highly regulated by a double-negative feedback loop consisting of TGF-β1/ZEB pathway and miR-200 family, which therefore has become a promising target for cancer chemotherapy. Pien Tze Huang (PZH), a well-known traditional Chinese formula first prescribed in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of various types of human malignancy including colorectal cancer (CRC). Our published data proposed that PZH was able to induce apoptosis, inhibit cell proliferation and tumor angiogenesis, leading to the suppression of CRC growth in vitro and in vivo. To further elucidate the mode of action of PZH, in the present study we evaluated its effects on the metastatic capacities of human colorectal carcinoma HCT-8 cells and investigated the underlying molecular mechanisms. We found that PZH significantly inhibited the migration and invasion of HCT-8 cells in a dose-dependent manner. In addition, PZH treatment inhibited the expression of key mediators of TGF-β1 signaling, such as TGF-β1, Smad2/3 and Smad4. Moreover, PZH treatment suppressed the expression of ZEB1 and ZEB2, two critical target genes of TGF-β1 pathway, leading to a decrease in the expression of mesenchymal marker N-cadherin and an increased expression of epithelial marker E-cadherin. Furthermore, PZH treatment upregulated the expression of miR-200a, miR-200b and miR-200c. Collectively, our findings in this study suggest that PZH can inhibit metastasis of colorectal cancer cells via modulating TGF-β1/ZEB/miR-200 signaling network, which might be one of the mechanisms whereby PZH exerts its anticancer function.

Smad-interacting protein-1 (SIP1), also known as deltaEF2, ZEB2 and zfhx1b, is essential for the formation of the neural tube and the somites. Overexpression of Xenopus SIP1 causes ectopic neural induction via inhibition of bone morphogenetic protein (BMP) signaling and inhibition of Xbra expression. Here, we report the functional analyses of 4 domain-deletion mutants of XSIP1. Deletion of the N-terminus zinc finger domain suppressed neural induction and BMP inhibition, but these were not affected by deletion of the other domains (the Smad binding domain, the DNA-binding homeodomain together with the CtBP binding site and the C-terminus zinc finger). Therefore SIP1 does not inhibit BMP signaling by binding to Smad proteins. In contrast, all of the deletion constructs inhibited Xbra expression. These results suggest that the N-terminus zinc finger domain of XSIP1 has an important role in neural induction and that Xbra suppression occurs via a mechanism separate from the neural inducing activity.