E26 Transformation Specific-1 Research Articles

The roles and mechanisms of ETS1 in autoimmune diseases and cancers: A comprehensive review

E26 transformation-specific-1 (ETS1) is a founding member of the ETS transcription factor family. This transcription factor is highly conserved in amino acid sequences and highly expressed in many immune tissues, such as the thymus, spleen, and lymph gland. ETS1 plays multiple regulatory roles in immune-related diseases and acts as a transcriptional activator or inhibitor of many genes to regulate immune cell differentiation, development, apoptosis, and tumor occurrence. The expression level of ETS1 is correlated with disease severity. However, the molecular mechanisms behind disease and tumor progression mediated by ETS1 have not been fully elucidated. In addition, the therapeutic potential of ETS1 in clinical treatment remains to be further explored. Here, we review and summarize the molecular structure and functions of ETS1 and then focus on the roles of ETS1 in the occurrence of autoimmune diseases and cancers. This review provides a reference corroborating ETS1 as a potential therapeutic target for immune-related diseases and cancers.

LncRNA MALAT1 knockdown inhibits the development of choroidal neovascularization

In the pathogenesis of age-related macular degeneration, long non-coding RNAs have become important regulators. This study aimed to investigate the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of choroidal neovascularization (CNV) and the underlying mechanisms. The in vivo and in vitro model of CNV was established using laser-induced mouse CNV model and human choroidal vascular endothelial cells (HCVECs) exposed to hypoxia respectively. We explore the role of MALAT1 in the pathogenesis of CNV by using the small interference RNA both in vivo and in vitro. MALAT1 expression was found to be upregulated in the retinal pigment epithelial-choroidal complexes. MALAT1 knockdown inhibited CNV development and leakage in vivo and decreased HCVECs proliferation, migration, and tube formation in vitro. MALAT1 performed the task as a miR-17-5p sponge to regulate the expression of vascular endothelial growth factor A (VEGFA) and E26 transformation specific-1 (ETS1). This study provides a new perspective on the pathogenesis of CNV and suggests that the axis MALAT/miR-17-5p/VEGFA or ETS1 may be an effective therapeutic target for CNV.

Hsa_circ_0005548 knockdown repairs OGD/R-induced damage in human brain microvascular endothelial cells via miR-362-3p/ETS1 axis

Background Ischemic stroke (IS) is a highly prevalent type of stroke with very high rates of disability and death. As the regulatory role of circular RNAs (circRNAs) in various diseases has been revealed, we constructed a stroke cell model to analyze the action mechanism of hsa_circ_0005548 in IS. Methods The abundance of hsa_circ_0005548, microRNA-362-3p (miR-362-3p) and E26 transformation specific-1 (ETS-1) were measured by real-time quantitative polymerase chain reaction (RT-qPCR) or western blot. We constructed an IS cell model in vitro by oxygen-glucose deprivation/reperfusion (OGD/R) treatment and analyzed cell proliferation, apoptosis and inflammatory response through the use of Cell Counting Kit-8 (CCK8), 5-ethynyl-2’-deoxyuridine (EdU), flow cytometry and Enzyme-linked immunosorbent assay (ELISA), respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were employed for the analysis of the relationship between miR-362-3p and hsa_circ_0005548 or ETS1. Results The higher abundance of hsa_circ_0005548 and ETS-1 and lower level of miR-362-3p were observed in human brain microvascular endothelial immortalized (HBMEC-IM) cells under OGD/R. Hsa_circ_0005548 downregulation mitigated OGD/R-induced HBMEC-IM cell injury. Mechanistically, hsa_circ_0005548 targeted miR-362-3p. MiR-362-3p knockdown reversed the effect of hsa_circ_0005548 silencing on OGD/R-induced HBMEC-IM cell injury. ETS1 was validated as a direct target of miR-362-3p, and miR-362-3p attenuated OGD/R-induced HBMEC-IM cell injury by ETS1. Moreover, hsa_circ_0005548 modulated ETS1 via miR-362-3p. Conclusion Hsa_circ_0005548 knockdown repairs OGD/R-induced HBMEC-IM cell damage via miR-362-3p/ETS1 axis.

FOXO1-miR-506 axis promotes chemosensitivity to temozolomide and suppresses invasiveness in glioblastoma through a feedback loop of FOXO1/miR-506/ETS1/FOXO1.

To explore the role of forkhead box protein O1 (FOXO1) in the progression of glioblastoma multiforme (GBM) and related drug resistance, we deciphered the roles of FOXO1 and miR-506 in proliferation, apoptosis, migration, invasion, autophagy, and temozolomide (TMZ) sensitivity in the U251 cell line using in vitro and in vivo experiments. Cell viability was tested by a cell counting kit-8 (CCK8) kit; migration and invasion were checked by the scratching assay; apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. The construction of plasmids and dual-luciferase reporter experiment were carried out to find the interaction site between FOXO1 and miR-506. Immunohistochemistry was done to check the protein level in tumors after the in vivo experiment. We found that the FOXO1-miR-506 axis suppresses GBM cell invasion and migration and promotes GBM chemosensitivity to TMZ, which was mediated by autophagy. FOXO1 upregulates miR-506 by binding to its promoter to enhance transcriptional activation. MiR-506 could downregulate E26 transformation-specific 1 (ETS1) expression by targeting its 3'-untranslated region (UTR). Interestingly, ETS1 promoted FOXO1 translocation from the nucleus to the cytosol and further suppressed the FOXO1-miR-506 axis in GBM cells. Consistently, both miR-506 inhibition and ETS1 overexpression could rescue FOXO1 overactivation-mediated TMZ chemosensitivity in mouse models. Our study demonstrated a negative feedback loop of FOXO1/miR-506/ETS1/FOXO1 in GBM in regulating invasiveness and chemosensitivity. Thus, the above axis might be a promising therapeutic target for GBM.

Circular RNA DHTKD1 targets miR‑338‑3p/ETS1 axis to regulate the inflammatory response in human bronchial epithelial cells.

Asthma is a chronic inflammatory airway disease and the airway epithelium is involved in airway inflammation and innate immunity. However, whether circular RNA (circRNA) is involved in the pathogenesis of asthma remains unclear. The present study aimed to determine the functions and molecular mechanisms of circRNA targeting dehydrogenase E1 (circDHTKD1) in the inflammation response of human bronchial epithelial cells. BEAS-2B cells were stimulated with lipopolysaccharide (LPS) to establish a model of in vitro airway inflammation. Cell viability was assessed using Cell Counting Kit-8 assay. CircDHTKD1 was characterised by nucleocytoplasmic isolation and Sanger sequencing. The RNA expression levels of circDHTKD1, microRNA (miR)-338-3p and potential ERK pathway downstream genes were evaluated by reverse transcription-quantitative polymerase chain reaction. Western blot analysis was performed to measure associated protein levels. The levels of inflammatory cytokines were detected by ELISA. The interaction between circDHTKD1 and miR-338-3p was confirmed by dual-luciferase reporter assay. circDHTKD1 expression was significantly upregulated by LPS treatment, whereas miR-338-3p expression was decreased. Furthermore, circDHTKD1 directly targeted miR-338-3p, which negatively regulated expression of E26 transformation specific-1 (ETS1). Inflammatory cytokine and ETS1 expression levels decreased following transfection with small interfering RNA targeting circDHTKD1 or miR-338-3p mimics. In addition, co-transfection with miR-338-3p inhibitor reversed the effects caused by circDHTKD1 knockdown. The knockdown of ETS1 in LPS-induced BEAS-2B cells resulted in decreased cytokine production and inhibition of the ERK signalling pathway. Overall, these results suggested that the knockdown of circDHTKD1 alleviated the LPS-induced production of inflammatory cytokines and activation of the ERK pathway in BEAS-2B cells through the miR-338-3p/ETS1 axis. In summary, circDHTKD1 exacerbated LPS-triggered inflammation responses in BEAS-2B cells by regulating ETS1 expression by interacting with miR-338-3p, suggesting that circDHTKD1 may serve as a potential therapeutic target against asthma.

DNA methyltransferase 1 (DNMT1) suppresses mitophagy and aggravates heart failure via the microRNA-152-3p/ETS1/RhoH axis

AbstractDNA methyltransferase 1 (DNMT1) shows close link with heart disease. This study aimed to define the role DNMT1 plays in heart failure and determine the underlying mechanism. Expression of microRNA (miR)-152-3p, DNMT1, E26 transformation specific-1 (ETS1) and ras homolog gene family member H (RhoH) was determined by RT-qPCR and/or western blot analysis. The interaction between miR-152-3p and ETS1 was predicted and verified. Methylation of the miR-152-3p promoter region was assessed using methylation-specific PCR. H9c2 cells were chosen for in vitro assays to examine the regulatory role of DNMT1 in autophagy and mitophagy with respect to miR-152-3p/ETS1/RhoH. Doxorubicin (DOX)-induced rat models of heart failure were employed for in vivo validation. DNMT1 expression was upregulated in the heart tissues of DOX-induced rats, where it showed an inverse correlation with miR-152-3p expression. Moreover, DNMT1 was shown to enhance methylation of the miR-152-3p promoter region and suppress its expression, leading to inhibition of mitophagy in H9c2 cells. In addition, DNMT1 enhanced expression of ETS1, which further elevated RhoH expression. Moreover, ETS1-elevated RhoH reduced cell viability and promoted autophagy and mitophagy in H9c2 cells upon treatment with DOX. Next, in vivo results demonstrated that depletion of DNMT1 protected rats from heart failure in a miR-152-3p/ETS1/RhoH-dependent manner. Overall, these findings indicate that DNMT1 may inhibit expression of miR-152-3p by promoting the methylation of miR-152-3p and enhancing the expression of ETS1, thereby inducing RHOH transcriptional activation and inhibiting mitochondrial autophagy, ultimately promoting the development of heart failure.

Endothelial Loss of ETS1 Impairs Coronary Vascular Development and Leads to Ventricular Non-Compaction.

Jacobsen syndrome is a rare chromosomal disorder caused by deletions in the long arm of human chromosome 11, resulting in multiple developmental defects including congenital heart defects. Combined studies in humans and genetically engineered mice implicate that loss of ETS1 (E26 transformation specific 1) is the cause of congenital heart defects in Jacobsen syndrome, but the underlying molecular and cellular mechanisms are unknown. To determine the role of ETS1 in heart development, specifically its roles in coronary endothelium and endocardium and the mechanisms by which loss of ETS1 causes coronary vascular defects and ventricular noncompaction. ETS1 global and endothelial-specific knockout mice were used. Phenotypic assessments, RNA sequencing, and chromatin immunoprecipitation analysis were performed together with expression analysis, immunofluorescence and RNAscope in situ hybridization to uncover phenotypic and transcriptomic changes in response to loss of ETS1. Loss of ETS1 in endothelial cells causes ventricular noncompaction, reproducing the phenotype arising from global deletion of ETS1. Endothelial-specific deletion of ETS1 decreased the levels of Alk1 (activin receptor-like kinase 1), Cldn5 (claudin 5), Sox18 (SRY-box transcription factor 18), Robo4 (roundabout guidance receptor 4), Esm1 (endothelial cell specific molecule 1) and Kdr (kinase insert domain receptor), 6 important angiogenesis-relevant genes in endothelial cells, causing a coronary vasculature developmental defect in association with decreased compact zone cardiomyocyte proliferation. Downregulation of ALK1 expression in endocardium due to the loss of ETS1, along with the upregulation of TGF (transforming growth factor)-β1 and TGF-β3, occurred with increased TGFBR2/TGFBR1/SMAD2 signaling and increased extracellular matrix expression in the trabecular layer, in association with increased trabecular cardiomyocyte proliferation. These results demonstrate the importance of endothelial and endocardial ETS1 in cardiac development. Delineation of the gene regulatory network involving ETS1 in heart development will enhance our understanding of the molecular mechanisms underlying ventricular and coronary vascular developmental defects and will lead to improved approaches for the treatment of patients with congenital heart disease.

Ets1 mediates sorafenib resistance by regulating mitochondrial ROS pathway in hepatocellular carcinoma

The incidence and mortality of hepatocellular carcinoma (HCC) are on a rise in the Western countries including US, attributed mostly to late detection. Sorafenib has been the first-line FDA-approved drug for advanced unresectable HCC for almost a decade, but with limited efficacy due to the development of resistance. More recently, several other multi-kinase inhibitors (lenvatinib, cabozantinib, regorafenib), human monoclonal antibody (ramucirumab), and immune checkpoint inhibitors (nivolumab, pembrolizumab) have been approved as systemic therapies. Despite this, the median survival of patients is not significantly increased. Understanding of the molecular mechanism(s) that govern HCC resistance is critically needed to increase efficacy of current drugs and to develop more efficacious ones in the future. Our studies with sorafenib-resistant (soraR) HCC cells using transcription factor RT2 Profiler PCR Arrays revealed an increase in E26 transformation–specific-1 (Ets-1) transcription factor in all soraR cells. HCC TMA studies showed an increase in Ets-1 expression in advanced HCC compared to the normal livers. Overexpression or knocking down Ets-1 modulated sorafenib resistance-related epithelial–mesenchymal transition (EMT), migration, and cell survival. In addition, the soraR cells showed a significant reduction of mitochondrial damage and mitochondrial reactive oxygen species (mROS) generation, which were antagonized by knocking down Ets-1 expression. More in-depth analysis identified GPX-2 as a downstream mediator of Ets-1-induced sorafenib resistance, which was down-regulated by Ets-1 knockdown while other antioxidant pathway genes were not affected. Interestingly, knocking down GPX2 expression significantly increased sorafenib sensitivity in the soraR cells. Our studies indicate the activation of a novel Ets-1–GPX2 signaling axis in soraR cells, targeting which might successfully antagonize resistance and increase efficacy.

E26 transformation-specific 1 is implicated in the inhibition of osteogenic differentiation induced by chronic high glucose by directly regulating Runx2 expression.

Chronic high glucose (HG) plays a crucial role in the pathogenesis of diabetes-induced osteoporosis by inhibiting the differentiation and proliferation of osteoblasts. This study aims to examine the role of E26 transformation-specific 1 (ETS1) in the inhibition of osteoblast differentiation and proliferation caused by chronic HG, as well as the underlying mechanism. Chronic HG treatment downregulated ETS1 expression and inhibited differentiation and proliferation of MC3T3-E1 cells. Downregulation of ETS1 expression inhibited the differentiation and proliferation of MC3T3-E1 cells under normal glucose conditions, and ETS1 overexpression attenuated the damage to cells exposed to chronic HG. In addition, ETS1 overexpression reversed the decrease in runt-related transcription factor 2 (Runx2) expression in MC3T3-E1 cells treated with chronic HG. Using chromatin immunoprecipitation (ChIP) and luciferase reporter assays, we confirmed that ETS1 directly bound to and increased the activity of the Runx2 promoter. In summary, our study suggested that ETS1 was involved in the inhibitory effect of chronic HG on osteogenic differentiation and proliferation and may be a potential therapeutic target for diabetes-induced osteoporosis.

Silencing of SBF2-AS1 inhibits cell growth and invasion by sponging microRNA-338-3p in serous ovarian carcinoma.

Long noncoding RNA SET-binding factor 2 (SBF2) antisense RNA 1 (AS1) is associated with the growth and metastasis of multiple cancer types, but its biological roles in serous ovarian carcinoma (SOC) remain unclear. In this study, the aberrant upregulation of SBF2-AS1 is detected in SOC after analysis of differentially expressed genes between SOC tissues and normal fallopian tubes from the public Gene Expression Omnibus (GEO) database. We determine that knockdown of SBF2-AS1 inhibits SOC cell proliferation and invasion by sponging miR-338-3p. MiR-338-3p acts as a tumor suppressor in SOC, and E26 transformation specific-1 (ETS1) is identified as a potential target of miR-338-3p regulation. Furthermore, SBF2-AS1 could modulate ETS1 by operating as a competing endogenous RNA for miR-338-3p. This finding elucidates a new mechanism for SBF2-AS1 in SOC development and provides a potential target for SOC therapeutic intervention.

LncRNA LINC00963 promotes osteogenic differentiation of hBMSCs and alleviates osteoporosis progression by targeting miRNA-760/ETS1 axis

Although long non-coding RNA LINC00963 has been reported to play a crucial regulatory role in osteoporosis (OP), its specific mechanism has not been well studied. Cell viability of human bone marrow mesenchymal stem cells (hBMSCs) transfected with short hairpin RNA targeting LINC00963 (sh-LINC00963) and negative control (sh-NC) was analysed by cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity in hBMSCs transfected with sh-LINC00963 and sh-NC after induction by osteogenic medium (OM) on day 7 was detected. The protein expression levels of osteocalcin (OCN) and osteopontin (OPN) in hBMSCs transfected with sh-LINC00963 and sh-NC during OM induction on day 3 were detected by western blot. The relationship among LINC00963, miR-760, and E26 transformation specific-1 (ETS1) was determined by bioinformatics analysis, luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) assay. A rat model with OP was established to confirm the role of LINC00963 in vivo. The expression level of LINC00963 was much lower in hBMSCs isolated from the discarded femoral head tissues of OP patients compared with that in health patients. Meanwhile, the expression level of LINC00963 was significantly increased and the expression level of miR-760 was decreased in hBMSCs during osteogenic induction. LINC00963 could bind to the 3′-untranslated region (3′-UTR) of miR-760 and negatively regulate the expression of miR-760, then promote the osteogenic differentiation in hBMSCs. ETS1 was identified as a target of miR-760. Moreover, overexpression of LINC00963 obviously reduced bone mineral density (BMD) of the left femur in OP rats and alleviated OP progression in vivo. Our results demonstrated that LINC00963 positively regulated the expression of ETS1 by directly targeting miR-760, and then promoted osteogenic differentiation of hBMSCs in vitro, and also attenuated OP progression in vivo, suggesting that LINC00963 might be a potential therapeutic target for OP.

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.

MALAT1 regulates osteogenic differentiation of human periodontal ligament stem cells through mediating miR-155-5p/ETS1 axis

BackgroundOsteogenic differentiation of human periodontal ligament stem cells is essential to periodontal regeneration treatment for periodontitis. This study investigated the mechanism of lncRNAs-related osteogenic differentiation. MethodsHuman periodontal ligament stem cells were extracted from human periodontal ligament and identified via flow cytometry. After being induced into osteogenic differentiation for three weeks using osteoblast inducing conditional media, human periodontal ligament stem cells were transfected with siRNA-MALAT1, or miR-155-5p inhibitor. Human periodontal ligament stem cells osteogenesis was observed by alkaline phosphatase staining, followed by alizarin red staining for evaluating mineralized nodes formation. Runt-related gene 2, collagen-1 and osteocalcin expressions were assessed by western blot and qRT-PCR. ResultsMALAT1 expression was assumed a negative correlation with miR-155-5p expression which was dropping over time in differentiating human periodontal ligament stem cells. MALAT1 could bind with miR-155-5p, and E26 transformation specific-1 (ETS1) was the targeted gene of miR-155-5p. Silenced MALAT1 suppressed ALP activity and mineralized nodes formation and inhibited the expressions of runt-related gene 2, collagen-1 and osteocalcin in differentiating human periodontal ligament stem cells, while miR-155-5p inhibitor reversed the effect of si-MALAT1 on differentiation of human periodontal ligament stem cells. ConclusionMALAT1 modulated differentiation of human periodontal ligament stem cells via regulating miR-155-5p.

ETS1 promoted cell growth, metastasis and epithelial-mesenchymal transition process in melanoma by regulating miR-16-mediated SOX4 expression.

Melanoma is a malignant tumor with high metastasis and mortality. Epithelial-mesenchymal transition (EMT) was reported to be involved in the growth and metastasis of melanoma. To investigate these sections further, we showed that E26 transformation specific 1 (ETS1) could regulate growth, metastasis and EMT process of melanoma by regulating microRNA(miR)-16/SRY-related HMG box (SOX) 4 expression. MiR-16, ETS1, SOX4 and nuclear factor κB (NF-κB) expression levels in melanoma cells were examined using qPCR. ETS1, SOX4, EMT-related proteins and NF-κB signaling pathway-related proteins were examined using western blot. Cell counting kit-8 assay, transwell assay were applied to evaluate the cell proliferation, migration and invasion of melanoma cells, respectively. Besides, a dual-luciferase reporter assay was employed to verify the binding relationship between ETS1 and miR-16, miR-16 and SOX4, miR-16 and NF-κB1. We showed that ETS1 and SOX4 were upregulated in melanoma cells, while miR-16 was downregulated. MiR-16 overexpression suppressed growth, metastasis and EMT process of melanoma. We found ETS1 could bind to the promoter region of miR-16 and inhibited its transcription. ETS1 silence could inhibit growth, metastasis and EMT process of melanoma, and inhibition of miR-16 could reverse the effects. Besides, miR-16 is directly bound to SOX4 and downregulated its expression. Rescued experiments confirmed that SOX4 overexpression abolished the inhibition effect of miR-16 mimics on growth, metastasis and EMT process of melanoma. Finally, NF-κB1 as the target of miR-16 mediated downstream biological responses. ETS1 activated NF-κB signaling pathway through miR-16 via targeting SOX4, thus promoting growth, metastasis and EMT of melanoma.

Bone marrow mesenchymal stem cell-derived exosomal MiR-338-3p represses progression of hepatocellular carcinoma by targeting ETS1.

The present study aims to explore the function of human bone marrow mesenchymal stem cell (BMSC)-derived exosomal micro ribonucleic acid (miR)-338-3p in hepatocellular carcinoma (HCC) and further investigate its effect on HCC cell functions. Firstly, BMSCs were co-cultured with HCC cells, and BMSC-derived exosomes were identified. Next, Transwell assay and methyl thiazolyl tetrazolium (MTT) experiments were carried out to detect the effects of miR-338-3p and E26 transformation specific-1 (ETS1) on the viability, invasion, migration, and apoptosis of HCC cells through the exosomes derived from BMSCs. Furthermore, the targeting relationship between miR-338-3p and EST1 was verified via bioinformatics study and dual-luciferase reporter gene analysis. Additionally, Western blotting (WB) was carried out to measure the expression levels of EST1 and other proteins in HCC cells. It was found that BMSCs inhibited HCC cell proliferation, invasion and migration, and induced cell apoptosis, while the inhibitors of exosomes played the opposite roles. In addition, the up-regulation of exosomal miR-338-3p or the silencing of EST1 restrained HCC cell proliferation, invasion and migration, and induced cell apoptosis. In conclusion, BMSC-derived exosomal miR-338-3p delays the development of HCC by down-regulating EST1, providing a new promising treatment target for HCC.

Expression of E26 transformation specific-1 (ETS-1) in tumour-infiltrating lymphocytes (TILs) is adverse prognostic factor in invasive breast cancer.

The microenvironment depicts the relationship between tumour cells and immune response, and every insight into stromal lymphocytes could contribute to explain their role and activity. E26 transformation specific-1 (ETS-1) is a transcription factor that is active in cell proliferation. We analysed its immunohistochemical expression in tumour infiltrating lymphocytes (TILs) in invasive breast cancer and correlated its immunohistochemical score (IHS) to traditional predictive and prognostic factors and survival. The sample contains data of 121 patients with invasive breast cancer, not otherwise specified (NOS) who underwent mammectomy and lymphadenectomy in 2002 at the Clinical Hospital Centre Zagreb, Croatia. Paraffin blocks of the tumour tissue were collected from the pathological archive. Three representative areas of every patient were chosen and multiple tissue samples were made. Immunohistochemical staining with rabbit anti-ETS-1 (Novocastra, UK) and the ABC method was performed on a DAKO Autostainer. The expression of ETS-1 in stromal TILs was analysed on an Olympus 41 microscope. The IHS score was calculated and correlated with clinical and pathological parameters, as well as disease-free survival (DFS) and overall survival (OS). In almost all patients (95%), some expression of ETS-1 in TILs was found. A moderate/high score of ETS-1 correlated with larger tumour size and higher histological grade, high proliferation index and low progesterone receptors (PgR). The patients with moderate/high ETS-1 expression in TILs had shorter DFS than patients with weak/negative ETS-1 expression. In invasive breast cancer NOS, expression of ETS-1 in TILs is an adverse prognostic factor.

MBRS-53. CONTROL OF MEDULLOBLASTOMA VASCULATURE BY A REGULATOR OF NEUROGENESIS

Medulloblastomas are characterized by poor neuronal lineage specification. Expression of the RE1 Silencing Transcription Factor (REST), a regulator of neurogenesis, is aberrantly elevated in human sonic hedgehog (SHH) medulloblastomas. Using a novel transgenic mouse (RESTTG) model, we demonstrated that REST is a driver of medulloblastoma genesis and promotes tumor progression in mice with loss of an allele of Ptch1 (Ptch+/−). Tumor formation in Ptch+/−/RESTTG mice occurred with 100% penetrance and a latency of 10–90 days in contrast to Ptch+/− mice, which developed tumors at a frequency of 15–20% at 6–9 months of age. Histopathological analyses showed leptomeningeal dissemination of tumors in Ptch+/−/RESTTG mice, in addition to a significant increase in tumor vasculature compared to tumors in Ptch+/− mice. These findings were recapitulated in xenografted tumors of isogenic low and high-REST medulloblastomas in mice. Proteome profiler human angiogenesis array analyses revealed a REST-dependent increase in vascular endothelial growth factor (VEGF) and placental growth factor (PLGF). Surprisingly, REST elevation also caused co-localization of tumor cells with tumor vasculature, specifically endothelial cells, and was associated with upregulated expression of a number of pro-angiogenic genes, including receptor VEGFR1 and the positive regulator of endothelial differentiation, E26 transformation specific-1 (ETS1), in tumor cells. In addition, expression of several anti-angiogenic molecules was downregulated. Knockdown of ETS1 reversed the above findings. Thus, our data demonstrate that REST elevation not only blocks neurogenesis in medulloblastoma cells, but also modulates the tumor microenvironment by mechanisms that likely involve vascular mimicry.

MiR-532-3p inhibits osteogenic differentiation in MC3T3-E1 cells by downregulating ETS1

BackgroundMany studies had identified that MicroRNAs (miRNAs) could affect bone metabolism by regulating the expression of various proteins. This study explored the effect and mechanism of miR-532-3p on osteogenic differentiation. MethodsWe analyzed the content of miR-532-3p in osteoporosis patients, osteoporosis rats, and osteogenic induced MC3T3-E1 cells. MiR-532-3p mimic or inhibitor utilized to alter intracellular miR-532-3p content. MTT method executed to detect the effect of miR-532-3p on osteoblast proliferation. Real-time qPCR, Western blot, alkaline phosphatase staining, and alizarin red staining utilized to ascertain the influence of miR-532-3p on osteogenic differentiation. Then, databases and a dual-luciferase reporter gene assay used to verify the target of miR-532-3p. Furthermore, the lentiviral vector was utilized to overexpress interesting target gene expression and checked whether the target gene was involved in the regulation of osteogenic differentiation by miR-532-3p. ResultsMiR-532-3p expression boosted in low bone mineral density (BMD) patients and rats. In MC3T3-E1 cells, miR-532-3p expression gradually decreased as osteogenic induction matures. MiR-532-3p mimic negatively regulated succinate dehydrogenase (SDH) activity, alkaline phosphatase (ALP) activity, mineralization ability, the osteogenic-associated gene (Col1A1, Runx2, ALP, OPN, and OCN) and E−26 transformation specific-1 (ETS1) expression of MC3T3-E1 cells. Things are the opposite of the miR-532-3p inhibitor. ETS1 identified as the miR-532-3p target gene, and miR-532-3p could inhibit its expression. Besides, improved ETS1 expression could rescue the suppressive effect of miR-532-3p mimic on osteogenic differentiation. ConclusionmiR-532-3p can suppress osteogenic differentiation by downregulating ETS1 expression.

MiR‑512‑5p suppresses proliferation, migration and invasion, and induces apoptosis in non‑small cell lung cancer cells by targeting ETS1.

An increasing number of microRNA (miRNA) have been demonstrated to serve as molecular biomarkers for tumor cell progression. miR-512-5p was revealed as oncogenic regulator in several types of cancer. However, whether and how miR-512-5p regulates non-small cell lung cancer (NSCLC) remains unclear. In the present study, the expression of miR-512-5p was detected in NSCLC tissues and cell lines. Then, the proliferation, migration, invasion and apoptosis in NSCLC A549 and H1299 cell lines were detected when miR-512-5p was overexpressed. Furthermore, the underlying mechanism was identified. The level of miR-512-5p was decreased in NSCLC tissues and in NSCLC cells compared with adjacent normal tissues and normal lung tissue cell lines. miR-512-5p mimics inhibited the cell proliferation, migration, invasion and induced apoptosis in A549 and H1299 cells. In addition, a luciferase reporter assay suggested that overexpression of miR-512-5p may decrease the expression of the E26 transformation specific-1 (ETS1) gene; it was subsequently verified that downregulation of the ETS1 gene inhibited cell proliferation and migration and induced cell apoptosis in A549 and H1299 cells, and ETS1 small interfering RNA in the presence of an miR-512-5p inhibitor reversed the effect. The data described in the present study suggest that miR-512-5p may be a tumor suppressor and a potential treatment target in NSCLC.

MicroRNA-338-3p inhibits the progression of bladder cancer through regulating ETS1 expression.

MicroRNA-338-3p (miR-338-3p) was reported to influence the metastasis and development of several human cancers. However, in bladder cancer (BC), the special function of miR-338-3p remains unknown. Here, we aimed at exploring the miR-338-3p function in the progression of BC. miR-338-3p and ETS1 expressions were examined by quantitative Real-time polymerase chain reaction (qRT-PCR) in BC samples. Following that, transwell assays for cell migration and invasion were performed. And MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for cell proliferation was conducted as well. Western blot was employed to examine the epithelial-mesenchymal transition (EMT) marker expressions. Finally, the relationship between miR-338-3p and E26 transformation specific-1 (ETS1) was verified by luciferase reporter assay. The decreased miR-338-3p expression was examined in BC cells. Moreover, miR-338-3p upregulation repressed cell proliferation ability in BC. Next, miR-338-3p upregulation also depressed cell metastasis and EMT in BC cells. Furthermore, ETS1 was a direct target of miR-338-3p and inversely associated with its expression. And upregulation of ETS1 partially rescued the suppression of miR-338-3p for cell proliferation and metastasis in BC. Upregulation of miR-338-3p inhibited the proliferation, metastasis and EMT in BC by suppressing ETS, showing that miR-338-3p might block the development of BC through regulating ETS1 expression.