406983

MIR200A

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133(+) side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by beta-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-beta-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-beta-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers.CI - (c)2011 AACR.

Although the epithelial-mesenchymal transition (EMT) is a normal process that occurs during development, it is thought to be associated with cancer progression and metastasis. Emerging evidence links mesenchymal stem cells (MSCs) in the tumor microenvironment with the occurrence of EMT in cancer progression. In this study, the human breast cancer cell line MCF7 was co-cultured with human adipose-derived MSCs (hAD-MSCs) in a transwell system. Co-cultured cells were analyzed for changes in cellular morphology, EMT markers, protein expression and tumor characteristics. We found that co-cultured MCF7 cells underwent EMT and established a stable mesenchymal phenotype after prolonged co-culturing. Here, we demonstrate that paracrine transforming growth factor-beta1 (TGF-beta1) secreted by hAD-MSCs regulated the establishment of EMT in MCF7 cells by targeting the ZEB/miR-200 regulatory loop. The downregulation of paracrine TGF-beta1 levels can inhibit and reverse the EMT progress by downregulating ZEB1/2 and upregulating miR-200b and miR-200c. The maintenance of a stable mesenchymal state by MCF7 cells required the establishment of autocrine TGF-beta signaling to drive and sustain ZEB expression, which had been initiated by the prolonged co-culturing with hAD-MSCs. These results suggest that MSCs may promote breast cancer metastasis by stimulating and facilitating the EMT process.

BACKGROUND: MicroRNAs (miRNAs) play an essential role in mediating gene expression in both normal and malignant cells. However, little is known about specific miRNAs during the development of hepatocellular carcinoma (HCC) from well-differentiated to poorly differentiated cells. METHODS: We performed miRNA array analysis of three different HCC cell lines: HepG2, HepJ5, and skHep-1. The expression patterns of miR-200 family members were confirmed by real-time polymerase chain reaction (PCR). We overexpressed miR-200 family members by using a lentivirus system and selected for stably transduced cells using antibiotics. The migration ability of the cells was tested using the Transwell migration assay system. RESULTS: Our miRNA array and real-time PCR results indicated a decrease in the expression of miR-200 family members in poorly differentiated skHep-1 cells compared with well-differentiated HepG2 cells. We overexpressed miR-200a and miR-200b in both HepJ5 and skHep-1 cells and found that the overexpression of the miR-200 family members did not influence proliferation, although migration was decreased in these cells. We found that overexpression of miR-200 family members led to an upregulation of E-cadherin expression in both HepJ5 and skHep-1 cells. Furthermore, we silenced E-cadherin expression by shRNA in miR200a-HepJ5 cells and found that the migratory ability of these cells was enhanced upon the decrease in E-cadherin expression. CONCLUSIONS: Members of the miR-200 family (miR-200a and miR-200b) play important roles in HCC migration by regulating E-cadherin expression.

Concepts and experimental models derived from basic research have been successfully applied to the field of molecular oncology, tremendously increasing our knowledge of the nature and the progression of tumors. The process of epithelial-to-mesenchymal transition, the cancer stem cell hypothesis, and their functional association and interdependence represent some of the most significant examples. The molecular determinants underlying the plasticity of cancers are currently the object of extensive research efforts, and a substantial body of evidence suggests that these models can be connected by the regulatory role of microRNAs, small noncoding RNA molecules with a fundamental role in many cellular functions. This review will highlight and discuss this link and its possible implications for the fight against cancer.

The differentiation of human pluripotent stem cells (hPSCs) to insulin-expressing beta islet-like cells is a promising in vitro model system for studying the molecular signaling pathways underlying beta cell differentiation, as well as a potential source of cells for the treatment of type 1 diabetes. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate many biological processes, including cellular differentiation. We studied the miRNA and mRNA expression profiles of hPSCs at five stages of in vitro differentiation along the pancreatic beta cell lineage (definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor and hormone-expressing endocrine cells) in the context of samples of primary human fetal pancreas and purified adult islet cells using microarray analysis. Bioinformatic analysis of the resulting data identified a unique miRNA signature in differentiated beta islet cells, and predicted the effects of key miRNAs on mRNA expression. Many of the predicted miRNA-mRNA interactions involved mRNAs known to play key roles in the epithelial-mesenchymal transition process and pancreatic differentiation. We validated a subset of the predictions using qRT-PCR, luciferase reporter assays and western blotting, including the known interaction between miR-200 and ZEB2 (involved in epithelial-mesenchymal transition) and the novel interaction between miR-200 and SOX17 (a key transcription factor in specification of definitive endoderm). In addition, we found that miR-30d and let-7e, two miRNAs induced during differentiation, regulated the expression of RFX6, a transcription factor that directs pancreatic islet formation. These findings suggest that precise control of target mRNA expression by miRNAs ensures proper lineage specification during pancreatic development.

BACKGROUND: Pancreatic cancer is one of the most aggressive cancers, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells. And miR-200 has been identified as a powerful regulator of EMT. METHODS: cancer Stem Cells (CSCs) of human pancreatic cancer cell line PANC-1 were processed for CD24, CD44 and ESA multi-colorstaining, and sorted out on a BD FACS Aria II machine. RT-qPCR was performed using the miScript PCR Kit to assay the expression of miR-200 family. In order to find the role of miR-200a in the process of EMT, miR-200a mimic was transfected to CSCs. RESULTS: Pancreatic cancer cells with EMT phenotype displayed stem-like cell features characterized by the expression of cell surface markers CD24, CD44 and epithelial-specific antigen (ESA), which was associated with decreased expression of miR-200a. Moreover, overexpression of miR-200a was resulted in down-regulation of N-cadherin, ZEB1 and vimentin, but up-regulation of E-cadherin. In addition, miR-200a overexpression inhibited cell migration and invasion in CSCs. CONCLUSION: In our study, we found that miR-200a played an important role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in pancreatic cancer. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to mesenchymal-to-epithelial transition (MET) phenotype using novel agents would be useful for prevention and/or treatment of pancreatic cancer.

Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial-to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was initiated to explore whether ZEB2 expression differs in prostate cancer (PCa, n=7) and benign prostatic hyperplasia (BPH, n=7) tissues. In PCa tissues, the levels of both immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher (P<0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P<0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P<0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection of AR and ZEB2 cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen-dependent cells and as a negative regulator in AI PCa cells.

Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor beta (TGFbeta)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFbeta-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFbeta-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.CI - (c)2014 American Association for cancer Research.

BACKGROUND: Hepatocellular carcinoma (HCC) typically relies on tumor transformation and angiogenesis for its malignant behavior, including growth and metastasis. Previously, we reported that Vasohibin2 (VASH2) is preferentially expressed in hepatocellular carcinoma (HCC) tumor tissues and promotes angiogenesis. Here, we further investigated the role of VASH2 in HCC tumor progression. RESULTS: Bioinformatics analyses and luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200a/b/c. We then used HepG2 and Hep3B cells, two representative hepatic cancer cell lines, to examine the role of VASH2 in tumors. VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT. Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway. CONCLUSION: VASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo. These results indicated that VASH2 expression in HCC cells promotes the malignant transformation of tumors by inducing EMT.

Immunosuppression of tumour-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8(+) TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumour cells, leading to CD8(+) T-cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T-cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

Due to high incidence of invasion and intrahepatic metastasis, hepatocellular carcinoma (HCC) is one of the most aggressive tumors in the world, which is also associated with the acquisition of epithelial-mesenchymal transition (EMT). Increasing evidence suggests that cancer cells with EMT traits share many biological characteristics with cancer stem cells. And miR-200a has been known as a powerful regulator of EMT. Here, we sought to investigate the role of miR-200a in regulation of EMT phenotype of liver cancer stem cells (LCSCs). We used side population (SP) sorting to obtain cancer stem-like cells from HCC cell lines and identified that the SP fraction could be enriched with LCSCs. Then, we detected the expression of miR-200a and EMT makers in SP and non-SP cells. Our results suggested that miR-200a was down-regulated in SP cells, along with relatively low epithelial marker and high mesenchymal marker. In order to find the role of miR-200a in the manipulation of EMT, we transfected miR-200a mimic into LCSCs and found that overexpression of miR-200a resulted in down-regulation of N-cadherin, ZEB2, and vimentin, but up-regulation of E-cadherin. Moreover, overexpression of miR-200a resulted in decreased migration and invasion ability in LCSCs. In conclusion, our study revealed that miR-200a played an important role in linking the characteristics of cancer stem cells with EMT phenotype in HCC, and targeting miR-200a might be an effective strategy to weaken the invasive behavior of LCSCs.

This study was set to study the molecular mechanism underlying how miR-200 regulates EGF/EGFR signaling to involve in epithelial-mesenchymal transition (EMT) in anaplastic thyroid cancer (ATC) cells. Loss-of-function experiments of EGFR silencing by siRNA transfection was performed. Transfection of pre-miR-200s or anti-miR-200s was used to increase or decrease miR-200 transcripts. Real-time PCR, Western blot, immunohistochemistry, and transwell experiments were performed to determine the role of miR-200s in EMT and its role in EGF/EGFR-mediated EMT in vitro and in vivo. EGF/EGFR signaling activation increased the expression of mesenchymal marker vimentin in Nthy-ori 3-1 cells and decreased the expression of endothelial maker E-cadherin. EGF stimulation led to increased RhoA expression in Nthy-ori 3-1 cells. EGFR silencing resulted in decreased RhoA expression in SW1736 and ARO cells. EGF stimulation led to down-regulation of miR-200s and EMT. Restoration of miR-200 expression by pre-miR-200a/c transfection reversed the process, including increased E-cadherin and decreased vimentin. Down-regulation of miR-200 by anti-miR-200 effectively reduced miR-200. Matrigel invasion assay proved that restoration of miR-200 expression counteracted invasiveness. EGFR silencing decreased invasiveness in SW1736 cells, while down-regulation of miR-200s restored invasiveness. Xenograft tumors of SW1736 cells with cotransfection of anti-miR-200s and EGFR siRNA which kept the similar E-cadherin and vimentin expression with the untransfected controls. In ATC cells, miR-200s play a central role in EGF/EGFR-mediated invasiveness in vitro and EMT in vivo.

BACKGROUND: Restin belongs to MAGE superfamily and is known as MAGE H1. Restin was firstly cloned from HL-60 cells treated with all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if Restin plays a role in breast cancer cells in vitro and in vivo. METHODS AND RESULTS: Real-time PCR and western blot were conducted to detect Restin expression in multiple breast cancer cell lines and Restin level was negatively related with cell motility. Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology, wound closure assay, transwell migration and invasion assays were performed to detect if Restin inhibited EMT. Our data showed that Restin overexpressed cells exhibited classical epithelial cell morphology, and Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion. tumor xenograft model was used to characterize the biological functions of Restin in vivo. We found that Restin overexpression led to reduced lung metastasis. Real-time PCR, western blot, luciferase assay and ChIP assay were performed to identify the potential targets of Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by Restin. Unexpectedly, Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated Restin interacted with p73, one of p53 family members, which contributed to Restin-mediated activation of mir-200a/b and suppression of ZEB1/2. CONCLUSIONS: Taken together, our results suggest that Restin inhibits EMT and tumor metastasis by controlling the expression of the tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between Restin, EMT and tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II proteins may exert a tumor suppressive effect in vivo.

OBJECTIVE: Our study investigated the role of microRNA (miR)-200a and its molecular targets in hepatocellular carcinoma (HCC) cells. METHODS: An inhibitor of miR-200a was transiently transfected into the hepatocellular carcinoma cell line, MHCC-97L. The effect of this transfection on mRNA levels of epithelial-mesenchymal transition (EMT)-related genes was measured by fluorescence-based quantitative real-time polymerase chain reaction (qRT-PCR). Further, protein levels of EMT-related genes, cell proliferation and apoptosis-related markers were assessed by Western blot analysis in these transfected cells. MTT and wound-healing assay were used to evaluate the proliferation and migration of MHCC-97L cells in presence and in absence of miR-200a inhibitor. RESULTS: Compared with miR-NC control group, qRT-PCR results in anti-miR-200a group revealed a significant reduction in the mRNA levels of E-cadherin, with a concomitant increasing in vimentin mRNA level (all P < 0.05). Western blot results showed higher E-cadherin and Caspase-3 protein expressions in anti-miR-200a group compared to miR-NC group (P < 0.05). In addition, vimentin and Ki-67 protein expression was found sharply decreased in anti-miR-200a group compared to miR-NC group (P < 0.05). Consistent with this, wound-healing and MTT assay showed that migration and proliferation capacity of MHCC-97L cells in anti-miR-200a group is significantly increased compared with miR-NC group (both P < 0.05). CONCLUSION: Our study reveals an important role of miR-200a in inhibiting EMT, proliferation and migration in HCC cells, suggesting the possibility of miR-200a-based therapeutics in HCC.

epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.

epithelial-to-mesenchymal transition (EMT) and its reverse - mesenchymal to epithelial transition (MET) - are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During EMT, epithelial cells lose cell-cell adhesion and gain migratory and invasive traits either partially or completely, leading to a hybrid epithelial/mesenchymal (hybrid E/M) or a mesenchymal phenotype respectively. mesenchymal cells move individually, but hybrid E/M cells migrate collectively as observed during gastrulation, wound healing, and the formation of tumor clusters detected as Circulating tumor Cells (CTCs). Typically, the hybrid E/M phenotype has largely been tacitly assumed to be transient and 'metastable'. Here, we identify certain 'phenotypic stability factors' (PSFs) such as GRHL2 that couple to the core EMT decision-making circuit (miR-200/ZEB) and stabilize hybrid E/M phenotype. Further, we show that H1975 lung cancer cells can display a stable hybrid E/M phenotype and migrate collectively, a behavior that is impaired by knockdown of GRHL2 and another previously identified PSF - OVOL. In addition, our computational model predicts that GRHL2 can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of these PSFs may be predictive of poor patient outcome. Finally, based on these specific examples, we deduce certain network motifs that can stabilize the hybrid E/M phenotype. Our results suggest that partial EMT, i.e. a hybrid E/M phenotype, need not be 'metastable', and strengthen the emerging notion that partial EMT, but not necessarily a complete EMT, is associated with aggressive tumor progression.

Fibrosis is an important feature of inflammatory bowel diseases (IBD), particularly Crohn's disease (CD), but its pathogenesis is poorly understood. To determine the postulated involvement of epithelial-mesenchymal transition (EMT) in the development of fibrosis in IBD, we analysed the expression profiles of the miR-200 family which has been shown to induce EMT in experimental models and various human diseases. We also analysed the expression of Snail and Slug, postulated targets of the investigated microRNAs. Ten patients with ulcerative colitis (UC) and 10 patients with CD who underwent colon resection were included. From each, two tissue samples were chosen (one with the most severely and one with the least affected or normal mucosa) for analysis of microRNAs expression using real-time polymerase chain reaction, and Snail and Slug expression using immunohistochemistry. We found significant down-regulation of all investigated microRNAs in CD, and of three investigated microRNAs in UC, in comparison to the normal or the least affected mucosa. Comparing UC and CD, four microRNAs were significantly more down-regulated in CD than in UC. Snail and Slug were expressed in the injured epithelium and occasionally in mesothelial cells and submesothelial fibroblasts. Our finding of down-regulation of the miR-200 family and up-regulation of transcription repressors Snail and Slug supports the postulated role of EMT in the pathogenesis of fibrosis in IBD. The described expression patterns are consistent with the notion that fibrosis does not occur only in CD but also in UC, being much more severe in CD.CI - (c) 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

MicroRNA (miRNA) expression profiles were examined in 3 groups of lung carcinomas that had been stratified by increases in AKT1 or AKT2 gene number. Microarray analysis using 2000 probes revealed 87 miRNAs that were up-regulated and 32 down-regulated miRNAs in carcinomas harboring amplification or high-level polysomy of the AKT1 (AKT1+), as well as 123 up-regulated and 83 down-regulated miRNAs in those of the AKT2 genes (AKT2+), in comparison with carcinomas harboring disomy of both (AKTd/d). In total, 182 miRNAs were up-regulated in AKT1+ or AKT2+, compared with AKTd/d. Among these, 28 miRNAs were up-regulated in both the AKT1+ and AKT2+ groups, with a log2 ratio between 1.02 and 3.71 relative to AKTd/d group, including all miR-200 family members. Quantitative real-time polymerase chain reaction showed that carcinomas exhibiting lymph vessel invasion had significantly lower expression of miR-200a (P=.0230) and miR-200b (P=.0168), regardless of the status of the AKT genes. Moreover, a detailed statistical analysis revealed that, in adenocarcinoma and in the early stage of carcinomas (pathologic stage I/II), expression of miR-200a was higher in the AKT2+ group compared with the AKT1+ group, and these differences were statistically significant (P=.0334 and P=.0239, respectively). However, the expression of miR-200a was not significantly correlated with the expression of its target, the zinc finger E-box-binding homeobox 1 (ZEB1; P=.3801) or E-cadherin (P=.2840), a marker of the epithelial-mesenchymal transition. These results suggest that AKT2 can regulate miR-200a in a histology- or stage-specific manner and that this regulation is independent of subsequent involvement of miR-200a in epithelial-mesenchymal transition.CI - Copyright (c) 2016 Elsevier Inc. All rights reserved.

Highly upregulated in liver cancer (HULC), a lncRNA that is considered a key molecule in human liver cancer, has recently been revealed to be involved in hepatocellular carcinoma (HCC) development and progression [1, 2]. It has been reported that HULC can promote tumor invasion and metastasis of HCC, but its function and mechanism of action in HCC have not been elucidated. In this study, we found that HULC was aberrantly up-regulated in HCC tissues and associated with TNM stage, intrahepatic metastases, HCC recurrence, and postoperative survival. HULC depletion inhibited the growth and metastasis of HCC cell lines in vitro and in vivo. Moreover, HULC contributes to ZEB1-induced epithelial-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis that plays a key role in cancer progression. This effect of ZEB1 was inhibited by HULC siRNA. We conclude that the HULC functioned as a competing endogenous RNA (ceRNA) to mediate EMT via up-regulating ZEB1. In this way, it sequesters the miR-200a-3p signaling pathway to facilitate HCC metastasis. HULC comes into play as an oncogene in HCC, acting mechanistically by inducing HCC cells to activate EMT. Such an effect promotes tumor progression and metastasis through the miR-200a-3p/ZEB1 signaling pathway. The identification of this novel pathway that links high expression levels of HULC with EMT in HCC cells may serve as the foundation for the development of novel anti-tumor therapeutics.

Long non-coding RNAs have been revealed to play important roles in the progression of hepatocellular carcinoma. However, the detailed mechanisms underlying their activities are not fully understood. Using microarray technology, a number of long non-coding RNAs were previously identified to be aberrantly expressed in hepatocellular carcinoma. In this study, one of these long non-coding RNAs, designated lncRNA-PE (lncRNA promotes epithelial-mesenchymal transition), was further explored to study its expression profile and function. A cohort of human hepatocellular carcinoma tissue samples combined with benign controls and established human hepatocellular carcinoma cell lines were examined for the expression of lncRNA-PE. The biological functions of lncRNA-PE were examined by wound-healing and Transwell assays, which revealed that lncRNA-PE promotes cell invasion and migration. By detecting the level of epithelial-mesenchymal transition markers, lncRNA-PE was revealed to promote epithelial-mesenchymal transition in hepatocellular carcinoma cells. Further study suggested that lncRNA-PE downregulated miR-200a/b by repressing the primary transcript expression, enhanced ZEB1 expression, and promoted epithelial-mesenchymal transition of hepatocellular carcinoma cells. All these data imply that lncRNA-PE might play an important role in hepatocellular carcinoma development via the miR-200a/b-ZEB1 pathway.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is a crucial event in the development of renal interstitial fibrosis (RIF). A growing body of evidence indicates that beta-catenin plays an important role in various types of fibrosis. Although members of the microRNA (miRNA)-200 family have been suggested to suppress EMT in cancer and fibrosis, the function of miRNA-200a in regulating the progression of RIF is unknown. We speculate that miRNA-200a may hinder this progression through the suppression of beta-catenin. METHODS: Transforming growth factor beta1 (TGF beta1) was used to induce EMT of proximal tubule epithelial (HK-2) cells in vitro, quantitative real time polymerase chain reaction (qPCR) and Western Blot analysis measured the miRNA-200a and beta-catenin expression. qPCR, Western Blot analysis, Migration Assay and cell immunofluorescence were applied to detect the influence of up- and downregulated miRNA-200a expression and beta-catenin siRNA on beta-catenin and EMT. Dual luciferase report plasmid (CTNNB1 UTR WT/MT) verifies the target relationship between miRNA-200a and CTNNB1 (beta-catenin gene). RESULTS: miRNA-200a is downregulated and beta-catenin is upregulated during TGF beta1-induced EMT. Upregulation of miRNA-200a inhibits beta-catenin and attenuates TGF beta1-induced EMT and cell migration, while its downregulation increases beta-catenin and induces EMT and HK-2 cell migration. Further, knocking down beta-catenin suppressed the EMT induced by miRNA-200a downregulation. miRNA-200a directly targets for CTNNB1. CONCLUSIONS: miRNA-200a inhibits TGF beta1-induced EMT by directly targeting beta-catenin in proximal tubule epithelial cells.CI - (c) 2017 S. Karger AG, Basel.

Colorectal cancer (CRC) is associated with significant morbidity and mortality as many patients are diagnosed with advanced stage disease. MicroRNAs are small, noncoding RNA molecules that have a major role in gene expression regulation and are dysregulated in CRC. The miR-200 family is involved in epithelial-mesenchymal transition (EMT). This systematic review describes the roles of the miR-200 family in EMT in CRC. A search of electronic databases (PubMed and Embase) was conducted between January 2000 and July 2017. Both in vitro and human studies reporting on the miR-200 family and CRC were included. Studies describing molecular pathways and the role of the miR-200 family in the diagnostic and therapeutic management of CRC were analyzed. Thirty-four studies (22 in vitro and 18 human studies) were included. miR-200 family expression is regulated epigenetically and via transcriptional factor regulation. In vitro studies show that transfection of miR-200 family members into chemo-resistant colon cancer cell lines results in improved chemo-sensitivity and epithelial phenotype restoration. There is intra-tumoral variability in the tissue expression of miR-200 family members with decreased expression at the invasive front. Clinical studies in CRC patients have shown decreased primary tumor tissue expression of miR-429, miR-200a and miR-200c may be associated with worse survival. Conversely, increased blood levels of miR-141, miR-200a and miR-200c may be associated with worse outcomes. The miR-200 family has a central role in EMT. The miR200 family has potential for both prognostic and therapeutic management of CRC.CI - (c) 2018 UICC.

Endometrial cancer is the most common gynecological cancer. epithelial-mesenchymal transition (EMT) plays a critical role in tumor invasion and metastasis, which limits the success of treatment. Here, we investigated the roles of forkhead box A2 (FOXA2) and microRNA-200a (miR-200a) in regulating the EMT of endometrial cancer cells RL95-2. Empty vector or FOXA2 was stably transfected into RL95-2 cells. MTT assay measured cell proliferation, apoptosis assay measured apoptosis, Transwell invasion assay measured cell invasion, and Western blot measured the protein expression of FOXA2, E-cadherin, and vimentin. ChIP assay determined the binding of FOXA2 to E-cadherin promoter. For miR-200a analysis, the cells with stable FOXA2 expression were transfected with miR-negative control or miR-200a. Forced expression of FOXA2 decreased the proliferation and invasion, and increased the apoptosis of RL95-2 cells. FOXA2 also affected the EMT-associated proteins: FOXA2 increased the protein expression of E-cadherin and decreased the expression of vimentin. Moreover, FOXA2 positively regulated the promoter of E-cadherin in RL95-2 cells. Luciferase reporter assay identified FOXA2 as a target of miR-200a, which negatively regulated FOXA2. Western blot results showed that overexpression of miR-200a decreased the expression of E-cadherin but increased the expression of vimentin in the endometrial cancer cells by downregulating FOXA2 expression. FOXA2 may act as a tumor suppressor and inhibit EMT of endometrial cancer cells. FOXA2 expression is controlled by miR-200a, which promotes EMT of the endometrial cancer cells.

Hepatic stellate cells (HSCs) have an important role in liver fibrosis. epithelialtomesenchymal transition (EMT), which is promoted by the Hedgehog (Hh) signaling pathway, is involved in the activation of HSCs. MicroRNAs (miRNAs/miRs) have been reported to be involved in the progression of liver fibrosis. A previous study indicated that the activation of HSCs was suppressed by miR200a via targeting transforming growth factorbeta2 and betacatenin. However, whether miR200a is able to regulate the EMT in HSCs has remained elusive. The present study revealed that miR200a was decreased in vitro and in vivo during liver fibrosis. Furthermore, miR200a overexpression resulted in the inhibition of proliferation, alphaSMA expression and extracellular matrix production of activated HSCs. Of note, miR200a overexpression reduced myofibroblastic markers, including alphaSMA, type I collagen and desmin, and increased the epithelial cell marker Ecadherin. These results were further confirmed by immunofluorescence staining. Further study showed that the expression of genes associated with Hh signaling, including Hhip, Shh and Gli1, were not affected by miR200a. However, Gli2, a downstream signaling protein of the Hh pathway, was inhibited by miR200a and confirmed as a target of miR200a using a dual luciferase reporter assay. In addition, the inhibition of the Hh pathway by miR200a resulted in an increase of BMP7 and Id2 as well as a reduction of Snai1 and S100A4. Collectively, the results of the present study demonstrated that miR200a suppressed the EMT process in HSCs, at least in part, via Gli2.

epithelial tumor cells transit to a mesenchymal state in response to extracellular cues, in a process known as epithelial-to-mesenchymal transition (EMT). The precise nature of these cues has not been fully defined, an important issue given that EMT is an early event in tumor metastasis. Here, we have found that a population of metastasis-prone mouse lung adenocarcinoma cells expresses Notch and Notch ligands and that the Notch ligand Jagged2 promotes metastasis. Mechanistically, Jagged2 was found to promote metastasis by increasing the expression of GATA-binding (Gata) factors, which suppressed expression of the microRNA-200 (miR-200) family of microRNAs that target the transcriptional repressors that drive EMT and thereby induced EMT. Reciprocally, miR-200 inhibited expression of Gata3, which reversed EMT and abrogated metastasis, suggesting that Gata3 and miR-200 are mutually inhibitory and have opposing effects on EMT and metastasis. Consistent with this, high levels of Gata3 expression correlated with EMT in primary tumors from 2 cohorts of lung adenocarcinoma patients. These findings reveal what we believe to be a novel Jagged2/miR-200-dependent pathway that mediates lung adenocarcinoma EMT and metastasis in mice and may have implications for the treatment of human epithelial tumors.

Most chronic kidney injuries inevitably progress to irreversible renal fibrosis. Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play pivotal roles in the process of renal fibrosis. However, a comprehensive understanding of the pathogenesis of renal scar formation and progression remains an urgent task for renal researchers. The endogenously produced microRNAs (miRNAs), proved to play important roles in gene regulation, probably regulate most genes involved in EMT. In this study, we applied microarray analysis to investigate the expression profiles of miRNA in murine interstitial fibrotic kidneys induced by unilateral ureteral obstruction (UUO). It was found that miR-200a and miR-141, two members of the miR-200 family, were downregulated at the early phase of UUO. In TGF-beta1-induced tubular EMT in vitro, it was also found that the members of the miR-200 family were downregulated in a Smad signaling-dependent manner. It was demonstrated that the miR-200 family was responsible for protecting tubular epithelial cells from mesenchymal transition by target suppression of zinc finger E-box-binding homeobox (ZEB) 1 and ZEB2, which are E-cadherin transcriptional repressors. The results suggest that downregulation of the miR-200 family initiates the dedifferentiation of renal tubules and progression of renal fibrosis, which might provide important targets for novel therapeutic strategies.

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFbeta receptor (TGFbetaR3) is required for TGFbeta2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFbetaR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFbetaR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFbetaR3-mediated EMT when stimulated by TGFbeta2 or BMP-2. The introduction of TGFbetaR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFbeta2 or BMP-2, results in EMT. TGFbetaR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFbeta2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFbetaR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFbetaR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFbetaR3-mediated endothelial cell EMT.CI - Copyright A(c) 2011. Published by Elsevier Inc.