Circ_0005875 sponges miR-502-5p to promote renal cell carcinoma progression through upregulating E26 transformation specific-1.

Increasing evidence has shown that circular RNAs (circRNAs) play critical roles in various cancers, including renal cell carcinoma (RCC). We aimed to explore the role and underlying mechanism of circ_0005875 in RCC. The expression levels of circ_0005875, microRNA-502-5p (miR-502-5p) and E26 transformation specific-1 (ETS1) mRNA were determined by quantitative real-time PCR. Cell proliferation was assessed by Cell Counting Kit-8, colony formation, and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Cell migration and invasion were monitored by wound healing assay and transwell assay, respectively. Flow cytometry analysis was applied to determine cell apoptosis and cell cycle distribution. Western blot assay was performed to measure the protein expression of CyclinD1 and ETS1. The interaction between miR-502-5p and circ_0005875 or ETS1 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft tumor model was established to confirm the role of circ_0005875 in vivo. Circ_0005875 and ETS1 were upregulated and miR-502-5p was downregulated in RCC tissues and cells. Knockdown of circ_0005875 suppressed RCC cell proliferation, migration and invasion, and induced apoptosis and cell cycle arrest. MiR-502-5p was a target of circ_0005875, and miR-502-5p inhibition reversed the inhibitory effects of circ_0005875 knockdown on the malignant behaviors of RCC cells. ETS1 was a direct target of miR-502-5p, and miR-502-5p exerted its anti-tumor role in RCC cells by targeting ETS1. Moreover, circ_0005875 knockdown decreased ETS1 expression by sponging miR-502-5p. Additionally, circ_0005875 depletion suppressed tumor growth in vivo. Circ_0005875 knockdown suppressed RCC progression by regulating miR-502-5p/ETS1 axis, which might provide a promising therapeutic target for RCC.

Integrin ß6 acts as an unfavorable prognostic indicator and promotes cellular malignant behaviors via ERK-ETS1 pathway in pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly cancers and is expected to become the second leading cause of cancer death by 2030. Despite extensive efforts to improve surgical treatment, limited progress has been made. Increasing evidence indicates that integrin ß6 plays a crucial role in carcinoma invasion and metastasis. However, the expression and role of ß6 in PDAC remain largely unknown. In the present study, we investigated the expression of ß6 in PDAC and its potential value as a prognostic factor and therapeutic target. ß6 upregulation was identified as an independent unfavorable prognostic indicator. Integrin ß6 markedly promoted the proliferation and invasion of pancreatic carcinoma cells and induced ETS1 phosphorylation in an ERK-dependent manner, leading to the upregulation of matrix metalloprotease-9, which is essential for ß6-mediated invasiveness of pancreatic carcinoma cells. Accordingly, small interfering RNA-mediated silencing of integrin ß6 markedly suppressed xenograft tumor growth in vivo. Taken together, our results suggest that integrin ß6 plays important roles in the progression of pancreatic carcinoma and contributes to reduced survival times, and may serve as a novel therapeutic target for the treatment of PDAC.

Deregulation of ETS1 and FLI1 contributes to the pathogenesis of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. DLBCL is a heterogeneous disease characterized by different genetic lesions. We herein report the functional characterization of a recurrent gain mapping on chromosome 11q24.3, found in 23% of 166 DLBCL cases analyzed. The transcription factors ETS1 and FLI1, located within the 11q24.3 region, had significantly higher expression in clinical samples carrying the gain. Functional studies on cell lines showed that ETS1 and FLI1 cooperate in sustaining DLBCL proliferation and viability and regulate genes involved in germinal center differentiation. Taken together, these data identify the 11q24.3 gain as a recurrent lesion in DLBCL leading to ETS1 and FLI1 deregulated expression, which can contribute to the pathogenesis of this disease.

Estrogen receptor α enhances the transcriptional activity of ETS-1 and promotes the proliferation, migration and invasion of neuroblastoma cell in a ligand dependent manner.

BACKGROUND: It is well known that estrogen receptor α (ERα) participates in the pathogenic progress of breast cancer, hepatocellular carcinoma and head and neck squamous cell carcinoma. In neuroblastoma cells and related cancer clinical specimens, moreover, the ectopic expression of ERα has been identified. However, the detailed function of ERα in the proliferation of neuroblastoma cell is yet unclear. METHODS: The transcriptional activity of ETS-1 (E26 transformation specific sequence 1) was measured by luciferase analysis. Western blot assays and Real-time RT-PCR were used to examine the expression of ERα, ETS-1 and its targeted genes. The protein-protein interaction between ERα and ETS-1 was determined by co-IP and GST-Pull down assays. The accumulation of ETS-1 in nuclear was detected by western blot assays, and the recruitment of ETS-1 to its targeted gene's promoter was tested by ChIP assays. Moreover, SH-SY5Y cells' proliferation, anchor-independent growth, migration and invasion were quantified using the MTT, soft agar or Trans-well assay, respectively. RESULTS: The transcriptional activity of ETS-1 was significantly increased following estrogen treatment, and this effect was related to ligand-mediated activation of ERα. The interaction between the ERα and ETS-1 was identified, and enhancement of ERα activation would up-regulate the ETS-1 transcription factor activity via modulating its cytoplasm/nucleus translocation and the recruitment of ETS-1 to its target gene's promoter. Furthermore, treatment of estrogen increased proliferation, migration and invasion of neuroblastoma cells, whereas the antagonist of ERα reduced those effects. CONCLUSIONS: In this study, we provided evidences that activation of ERα promoted neuroblastoma cells proliferation and up-regulated the transcriptional activity of ETS-1. By investigating the role of ERα in the ETS-1 activity regulation, we demonstrated that ERα may be a novel ETS-1 co-activator and thus a potential therapeutic target in human neuroblastoma treatment.

Association of CXCR1 and 2 expressions with gastric cancer metastasis in ex vivo and tumor cell invasion in vitro.

BACKGROUND: CXCR1 and CXCR2, cell surface receptors of interleukin-8, regulate cell migration and alteration of their expression has been associated with poor prognosis of various cancers. The aim of this study was to detect their expression in gastric cancer to identify associations with another cell adhesion molecule, matrix metalloproteinase-9 (MMP9), and with clinicopathological data ex vivo, and then explore their potential role in gastric cancer cells in vitro. MATERIALS AND METHODS: A total of 172 cases of gastric cancer tissue specimens were collected for immunohistochemical analysis of CXCR1, CXCR2, and MMP9 expression. Expression of CXCR1 and CXCR2 proteins was knocked in or down using their cDNA and shRNA, respectively, in gastric cancer cell lines to assess the changed cell phenotypes and gene expression. RESULTS: CXCR1, CXCR2, and MMP9 were expressed in 61.0%, 77.9%, and 75.6% of gastric cancer tissues, respectively. Moreover, CXCR1 and CXCR2 expression was associated with tumor differentiations, advanced clinical stages, lymph node, and distant metastasis of gastric cancer. Similarly, MMP9 expression was associated with CXCR1 and CXCR2. Expression of these three proteins was interrelated. In vitro study showed that levels of CXCR1 and CXCR2 proteins were associated with the capacity of gastric cancer cell migration, while knockdown of their expression inhibited gastric cancer cell migration and invasion abilities in vitro. In contrast, overexpression of CXCR1 and CXCR2 proteins promoted tumor cell migration and invasion. At the gene levels, knockdown of CXCR1 or CXCR2 expression suppressed expression of Ets-1, SRC-1, and JNK proteins and phosphorylated c-Jun and Erk1/2. Conversely, upregulation of CXCR1 or CXCR2 promoted expression of Ets-1, SRC-1, JNK, and c-Jun proteins and phosphorylated JNK, c-Jun and Erk1/2. CONCLUSIONS: These findings suggest that CXCR1 and CXCR2 play an important role in gastric cancer progression. Further study will be performed to investigate whether target of their expression can be used as a novel strategy in clinical control of gastric cancer metastasis.

HBx and HBs regulate RhoC expression by upregulating transcription factor Ets-1.

The available evidence suggests that HBV proteins play an important role in the development of hepatocellular carcinoma (HCC). RhoC, a member of the Rho subfamily of the Ras superfamily of homologous genes, had been implicated in tumorigenesis and tumor progression. In a previous study, we demonstrated that HBx and HBs could up-regulate RhoC expression by enhancing its promoter activity. However, the specific mechanisms remain unclear. Here, we demonstrate that overexpression of Ets-1 results in upregulation of RhoC promoter activity and mRNA and protein levels. Expression of transcription factor Ets-1 was significantly higher in HepG2.2.15 cells than that in HepG2 cells. Meanwhile, infection of HepG2 cells with an HBV-adenovirus recombinant virus led to up-regulation of Ets-1. Of the four HBV proteins, HBx and HBs, could increase expression of Ets-1, which consequently contributed to the upregulation of RhoC. These findings might provide a novel insight into HBV-induced HCC metastasis.

ETS1 transcriptional activity is increased in advanced prostate cancer and promotes the castrate-resistant phenotype.

Advanced disease accounts for the majority of prostate cancer-related deaths and androgen deprivation therapy (ADT) is the standard of care for these patients. Many patients undergoing ADT become resistant to its effects and progress to castrate-resistant prostate cancer (CRPC). Current therapies for CRPC patients are inadequate, with progression-free survival rates as low as 2 months. The molecular events that promote CRPC are poorly understood. ETS (v-ets erythroblastosis virus E26 oncogene) transcription factors are regulators of carcinogenesis. Protein levels of the archetypical ETS factor, ETS1, are increased in clinical and latent prostate cancer relative to benign prostatic hyperplasia and normal prostate to promote multiple cancer-associated processes, such as energy metabolism, matrix degradation, survival, angiogenesis, migration and invasion. Our studies have found that ETS1 expression is highest in high-grade prostate cancer (Gleason 7 and above). Increased ETS1 expression and transcriptional activity promotes an aggressive and castrate-resistant phenotype in immortalized prostate cancer cells. Elevated AKT (v-akt murine thymoma viral oncogene homolog) activity was demonstrated to increase ETS1 protein levels specifically in castrate-resistant cells and exogenous ETS1 expression was sufficient to rescue invasive potential decreased by inhibition of AKT activity. Significantly, targeted androgen receptor activity altered ETS1 expression, which in turn altered the castrate-resistant phenotype. These data suggest a role for oncogenic ETS1 transcriptional activity in promoting aggressive prostate cancer and the castrate-resistant phenotype.

miR-200b targets Ets-1 and is down-regulated by hypoxia to induce angiogenic response of endothelial cells.

The miR-200 family plays a crucial role in epithelial to mesenchymal transition via controlling cell migration and polarity. We hypothesized that miR-200b, one miR-200 family member, could regulate angiogenic responses via modulating endothelial cell migration. Delivery of the miR-200b mimic in human microvascular endothelial cells (HMECs) suppressed the angiogenic response, whereas miR-200b-depleted HMECs exhibited elevated angiogenesis in vitro, as evidenced by Matrigel® tube formation and cell migration. Using in silico studies, miR target reporter assay, and Western blot analysis revealed that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), a crucial angiogenesis-related transcription factor, serves as a novel direct target of miR-200b. Knocking down endogenous Ets-1 simulated an anti-angiogenic response of the miR-200b mimic-transfected cells. Certain Ets-1-associated genes, namely matrix metalloproteinase 1 and vascular endothelial growth factor receptor 2, were negatively regulated by miR-200b. Overexpression of Ets-1 rescued miR-200b-dependent impairment in angiogenic response and suppression of Ets-1-associated gene expression. Both hypoxia as well as HIF-1α stabilization inhibited miR-200b expression and elevated Ets-1 expression. Experiments to identify how miR-200b modulates angiogenesis under a low oxygen environment illustrated that hypoxia-induced miR-200b down-regulation de-repressed Ets-1 expression to promote angiogenesis. This study provides the first evidence that hypoxia-sensitive miR-200b is involved in induction of angiogenesis via directly targeting Ets-1 in HMECs.

ETS1 promotes chemoresistance and invasion of paclitaxel-resistant, hormone-refractory PC3 prostate cancer cells by up-regulating MDR1 and MMP9 expression.

ETS1, which belongs to the ETS transcription factor family, plays important roles in diverse aspects of cancer such as drug resistance and metastasis. In the present study, we examined the functional roles of ETS1 in paclitaxel resistance and invasion using human prostate cancer PC3 cells and paclitaxel-resistant PC3PR cells established from PC3 cells. Our results showed that ETS1mRNA and protein expression was markedly up-regulated in paclitaxel-resistant PC3PR cells compared with paclitaxel-sensitive PC3 cells. The mRNA levels of MDR1 as well as MMP1, MMP3, MMP9 and uPA were positively correlated with that of ETS1. In PC3PR cells, silencing of ETS1 expression by siRNAs inhibited the activity of the MDR1 promoter containing ETS binding sites, reduced the mRNA and protein levels of MDR1 and suppressed paclitaxel resistance. Furthermore, ETS1 knockdown decreased secretion of MMP9 as well as its intracellular mRNA level, and dramatically inhibited invasion of PC3PR cells. Our results suggest that ETS1 promotes paclitaxel resistance and invasion in part by up-regulating MDR1 and MMP9 expression. Taken together, a novel therapeutic strategy targeting the ETS1 gene could be designed to overcome chemoresistance and metastasis of taxane-resistant, hormone-refractory prostate cancer.

Increased expression of IRF4 and ETS1 in CD4+ cells from patients with intermittent allergic rhinitis.

BACKGROUND: The transcription factor (TF) IRF4 is involved in the regulation of Th1, Th2, Th9, and Th17 cells, and animal studies have indicated an important role in allergy. However, IRF4 and its target genes have not been examined in human allergy. METHODS: IRF4 and its target genes were examined in allergen-challenged CD4(+) cells from patients with IAR, using combined gene expression microarrays and chromatin immunoprecipitation chips (ChIP-chips), computational target prediction, and RNAi knockdowns. RESULTS: IRF4 increased in allergen-challenged CD4(+) cells from patients with IAR, and functional studies supported its role in Th2 cell activation. IRF4 ChIP-chip showed that IRF4 regulated a large number of genes relevant to Th cell differentiation. However, neither Th1 nor Th2 cytokines were the direct targets of IRF4. To examine whether IRF4 induced Th2 cytokines via one or more downstream TFs, we combined gene expression microarrays, ChIP-chips, and computational target prediction and found a putative intermediary TF, namely ETS1 in allergen-challenged CD4(+) cells from allergic patients. ETS1 increased significantly in allergen-challenged CD4(+) cells from patients compared to controls. Gene expression microarrays before and after ETS1 RNAi knockdown showed that ETS1 induced Th2 cytokines as well as disease-related pathways. CONCLUSIONS: Increased expression of IRF4 in allergen-challenged CD4(+) cells from patients with intermittent allergic rhinitis leads to activation of a complex transcriptional program, including Th2 cytokines.

Phylogenetic and functional analysis identifies Ets-1 as a novel regulator of the Th2 cytokine gene locus.

The Th2 cytokine gene locus has emerged as a remarkable example of coordinated gene expression, the regulation of which seems to be rooted in an extensive array of cis-regulatory regions. Using a hypothesis-generating computational approach that integrated multispecies (n = 11) sequence comparisons with algorithm-based transcription factor binding-site predictions, we sought to identify evolutionarily conserved noncoding regions (ECRs) and motifs shared among them, which may underlie coregulation. Twenty-two transcription factor families were predicted to have binding sites in at least two Th2 ECRs. The ranking of these shared motifs according to their distribution and relative frequency pointed to a regulatory hierarchy among the transcription factor families. GATA sites were the most prevalent and widely distributed, consistent with the known role of GATA3 as a Th2 master switch. Unexpectedly, sites for ETS-domain proteins were also predicted within several Th2 ECRs and the majority of these sites were found to support Ets-1 binding in vitro and in vivo. Of note, the expression of all three Th2 cytokines (IL-5, -13, and -4) was significantly and selectively decreased in Th2 cells generated from Ets-1-deficient mice. Collectively, these data suggest that Ets-1 contributes to Th2 cytokine gene regulation by interacting with multiple cis-regulatory regions throughout the Th2 locus.

Purification, crystallization and preliminary X-ray crystallographic analysis of the ETS domain of human Ergp55 in complex with the cfos promoter DNA sequence.

The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS-cfos DNA complex crystal at a wavelength of 0.9725 Šon the BM14 synchrotron beamline at the ESRF, France. The ETS-cfos DNA complex crystal belonged to space group C222(1), with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS-cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein (PDB entry 1fli) and cfos DNA (PDB entry 1bc7) as search models. Structure analysis of the ETS-cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55.

p53 Deficiency leads to compensatory up-regulation of p16INK4a.

p53-p21-cyclin-dependent kinase and p16(INK4a)-cyclin-dependent kinase pathways have parallel functions in preventing tumorigenesis. In cancer patients, tumor suppressor p53 is frequently inactivated through mutations, whereas p16(INK4a) is silenced through promoter methylation. However, the interaction between these two pathways is less well understood. Here, we report that p53 controls p16(INK4a) expression in a unique way. p53 deficiency led to up-regulation of p16(INK4a) in primary mouse embryonic fibroblasts, osteoblasts, and various mouse organs, and an increase in the p16(INK4a) promoter activity, without affecting the half-life of p16(INK4a). Reconstitution of p53, but not mutant p53, restored the proper expression of p16(INK4a). These results indicate that p53 is necessary in repressing p16(INK4a) expression. However, up-regulation of p53 in response to genotoxic stress or nutlin-3 treatment did not down-regulate p16(INK4a). p53 did not repress the p16(INK4a) promoter activity either. These findings suggest that p53 has a necessary but not sufficient role in repressing p16(INK4a) expression. p16(INK4a) elevation in p53(-/-) cells is, at least partially, mediated by Ets1, a known positive regulator of p16(INK4a), as p53 deficiency up-regulated Ets1 through protein stabilization and knockdown of Ets1 down-regulated p16(INK4a) expression in p53(-/-) mouse embryonic fibroblasts. These studies uncover a compensatory mechanism for the loss of p53 and provide a basis for targeting both p53 and p16(INK4a) in cancer therapy.

Regulation of alphaB-crystallin gene expression by the transcription factor Ets1 in breast cancer.

Recent studies indicate that the small heat shock protein alphaB-crystallin is expressed in poor prognosis basal-like breast tumors and likely contributes to their aggressive phenotype. However, the mechanisms underlying the deregulated expression of alphaB-crystallin in basal-like tumors are poorly understood. Using a bioinformatics approach, we identified a putative DNA binding motif in the human alphaB-crystallin promoter for the proto-oncogene Ets1, a member of the ETS transcription factor family that bind to DNA at palindromic ETS-binding sites (EBS). Here we demonstrate that ectopic expression of Ets1 activates the alphaB-crystallin promoter by an EBS-dependent mechanism and increases alphaB-crystallin protein levels, while silencing Ets1 reduces alphaB-crystallin promoter activity and protein levels. Chromatin immunoprecipitation analyses showed that endogenous Ets1 binds to the alphaB-crystallin promoter in basal-like breast cancer cells in vivo. Interrogation of publically available gene expression data revealed that Ets1 is expressed in human basal-like breast tumors and is associated with poor survival. Collectively, our results point to a previously unrecognized link between the oncogenic transcription factor Ets1 and alphaB-crystallin in basal-like breast cancer.

TERT expression is susceptible to BRAF and ETS-factor inhibition in BRAFV600E/TERT promoter double-mutated glioma.

The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAFV600E and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAFV600E-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAFV600E-mutated HGG as compared to BRAFV600E-mutated LGG. In vitro, BRAFV600E/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAFV600E-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAFV600E and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.

Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: a useful tool for studying interactions between Ets-1 and its partners.

Identification of Ets-1 interaction partners is critical for understanding its properties. Ets-1 DNA-binding is governed by an intramolecular mechanism called autoinhibition. Ets-1 increases its DNA-binding affinity by counteracting autoinhibition through binding either to a particular organization of Ets binding sites (EBS) in palindrome, as in the Stromelysin-1 promoter, or to EBS adjacent to DNA-binding sites of its partners by combinatorial interactions, as in the Collagenase-1 promoter. Identification of new Ets-1 interaction partners should allow the identification of new functions for this transcription factor. To this end, we fused a biotin tag to Ets-1 protein in order to copurify it and its partners by affinity. For the first time, we cloned, produced in Escherichia coli and purified a biotinylated recombinant Ets-1 protein using the T7-Impact system (New England Biolabs), adapted to induce biotinylation. Nearly 100% biotinylation was attained without altering Ets-1 properties. Biotinylated Ets-1 bound to and transactivated the Stromelysin-1 promoter the same way as native Ets-1 did. It also conserved interactions with known Ets-1 partners such as c-Jun, Erk-2 and Runx-1. In addition, streptavidin pull-down and surface plasmon resonance assays demonstrated that biotinylated Ets-1 is a useful tool for qualitative and quantitative studies of Ets-1 interaction with its partners.

Mechanical strain induces osteogenic differentiation: Cbfa1 and Ets-1 expression in stretched rat mesenchymal stem cells.

Distraction osteogenesis is an active process of bone regeneration under controlled mechanical stimulation. Osteogenic differentiation of mesenchymal stem cells (MSCs) is essential for bone formation during this process. Cbfa1 and Ets-1 (core binding factor alpha 1 and v-ets erythroblastosis virus E26 oncogene homolog 1) are transcription factors that play important roles in the differentiation of MSCs to osteoblasts. In order to mimic a single activation of a clinical distraction device, a short period of cyclic mechanical strain (40 min and 2,000 microstrains) was applied to rat MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity were examined. The mRNA expression of Cbfa1 and Ets-1, as well as ALP, a specific osteoblast marker, was detected using real-time quantitative reverse transcription polymerase chain reaction. The results showed that mechanical strain can promote MSC proliferation, increase ALP activity and up-regulate the expression of Cbfa1 and Ets-1. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression was elevated later. The temporal expression pattern of ALP coincided perfectly with that of Cbfa1. Mechanical strain may act as a stimulator to induce differentiation of mesenchymal stem cells into osteoblasts, which is vital for bone formation in distraction osteogenesis.

Hepatocyte growth factor and inducible nitric oxide synthase are involved in multidrug resistance-induced angiogenesis in hepatocellular carcinoma cell lines.

Based on literature, it is possible to hypothesize that multidrug resistance (MDR) and angiogenic phenotypes are linked to each other in human liver cancer cells. Our goal is to assess whether MDR cells trigger angiogenesis and to study the possible molecular mechanisms involved. Conditioned medium from parental drug-sensitive P5 cells (P5-CM) and MDR-positive P1(0.5) cells [P1(0.5)-CM] stimulated human umbilical vein endothelial cells (HUVEC) survival, proliferation, migration, and microtubular structure formation, but P1(0.5)-CM had a significantly greater effect than P5-CM. Cell implants were done in the rabbit avascular cornea to measure angiogenesis in vivo: P1(0.5) cells induced an important neovascular response in rabbit cornea after 1 week, whereas P5 cells had no effect. P1(0.5) and P5 cells produced vascular endothelial growth factor, but only P1(0.5) secreted hepatocyte growth factor (HGF) into the medium, and small interfering RNA specific for MDR1 clearly reduced HGF production in P1(0.5) cells. The transcription factor Ets-1 and the HGF receptor c-Met were up-regulated in P1(0.5) cells and in HUVEC cultured in P1(0.5)-CM. Inducible nitric oxide synthase (iNOS) seemed to play a major role in the proangiogenic effect of P1(0.5), and its inhibition by 1400W blunted the capacity of P1(0.5) cells to stimulate HUVEC proliferation, migration, and Ets-1 expression. In conclusion, these data show that development of MDR and angiogenic phenotypes are linked to each other in MDR cells. HGF production, Ets-1 and c-Met up-regulation, and iNOS expression can be part of the molecular mechanisms that enhance the angiogenic activity of the MDR-positive hepatocellular carcinoma cell line.

miR-125a-5p Modulates Phenotypic Switch of Vascular Smooth Muscle Cells by Targeting ETS-1.

MicroRNAs are key regulators of vascular smooth muscle cells (VSMCs) phenotypic switch, one of the main events responsible for bare metal in-stent restenosis after percutaneous coronary intervention. miR-125a-5p is an important modulator of differentiation, proliferation, and migration in different cell types; however, its role in VSMCs is still unknown. The aim of this study was to evaluate the role of miR-125a-5p in VSMCs phenotypic switch. Our results suggest that miR-125a-5p is highly expressed in VSMCs, but it is down-regulated after vascular injury in vivo. Its overexpression is sufficient to reduce VSMCs proliferation and migration, and it is able to promote the expression of selective VSMCs markers such as alpha smooth muscle actin, myosin heavy chain 11, and smooth muscle 22 alpha. Interestingly, miR-125a-5p directly targets ETS-1, a transcription factor implicated in cell proliferation and migration and is crucial in PDGF-BB pathway in VSMCs. Thus, miR-125a-5p in this context inhibits PDGF-BB pathway and is therefore a potential regulator of VSMCs phenotypic switch.

ETS-1: A potential target of glycolysis for metabolic therapy by regulating glucose metabolism in pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies of all types of cancer due to lack of early symptoms and its resistance to conventional therapy. In our previous study, we have shown that v­ets erythroblastosis virus E26 oncogene homolog­1 (ETS­1) promote cell migration and invasion in pancreatic cancer cells. However, the function of ETS­1 in regulation of glycolysis and autophagy during progression of pancreatic cancer has not been defined yet. In this study, we sought to identify the potential role for silencing ETS­1 in reducing the expression of glucose transporter­1 (GLUT­1) to disturb glycolysis through alteration of 'Warburg effect', by which could result in AMP­activated protein kinase (AMPK) activation, autophagy induction and reduction of cell viability. MTT assay was applied to assess the cell viability in ETS­1 silencing cells and control groups. Glucose absorption rate, lactate production rate and cellular ATP level were measured by standard colorimetric assay kits. The levels of mRNAs of ETS­1, GLUT­1, autophagy­related gene 5 (ATG5) and ATG7 were analyzed by qRT­PCR. The expression of ETS­1, GLUT­1, ATG5, ATG7, p­AMPK, and LC3II proteins were evaluated by western blot analysis. GraphPad Prism 5.0 was used for all statistical analysis. We found that cell viability was obviously attenuated after silencing ETS­1. Besides, our results also showed that the expression of GLUT­1 significantly declined in ETS­1 silencing cell lines which resulted in a lower glucose utilization and lactate production. Furthermore, the inhibition of glycolysis, which depends on glucose utilization and lactate production, reduced the generation of energy in the form of ATP. Moreover, the reduction of cellular ATP was associated with stimulation of AMP­activated protein kinase (AMPK) and induction of autophagy. Our results indicated that ETS­1 induced autophagy after inhibition of glycolysis, and thus led to comparative decrease of cell viability. These results implied that ETS­1 could be a potential target for tumor metabolic therapy.