Enhancer Of Zeste Homolog 2 mRNA Research Articles

EZH2 Promotes Corneal Endothelial Cell Apoptosis by Mediating H3K27me3 and Inhibiting HO-1 Transcription

Purpose This paper aims to explore the molecular mechanism of Enhancer of Zeste Homolog 2 (EZH2)-mediated H3K27me3 in human corneal endothelial cells (HCEC) apoptosis by inhibiting Heme oxygenase-1 (HO-1) transcription to provide a potential target for the treatment of corneal apoptosis. Methods HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved-caspase-3) in HCECs; CCK-8 assay was conducted to detect cell viability and flow cytometry to analyze the apoptosis. HO-1 mRNA levels were detected by RT-qPCR and changes in H3K27me3 levels on the HO-1 promoter were detected by chromatin immunoprecipitation. Results HCECs transfected with si-EZH2 showed significantly lower EZH2 mRNA and protein levels, higher HCEC viability, lower apoptosis rates, higher antiapoptotic protein Bcl-2 expression, lower proapoptotic protein (Bax and Cleaved-caspase-3) levels, and significantly higher HO-1 expression. HCECs transfected with pcDNA3.1-EZH2 showed the opposite results. EZH2 repressed HO-1 transcription by mediating H3K27me3. H3K27me27 was enriched in the HO-1 promoter and overexpression of EZH2 increased H3K27me27 levels. Promotion of H3K27me3 partially reversed the mitigating effect of si-EZH2 on HCEC apoptosis. Overexpression of HO-1 partially reversed the apoptosis-promoting effects of EZH2 and H3K27me3 on HCECs. Conclusions EZH2 promotes HCE cell apoptosis by mediating H3K27me3 to inhibit HO-1 transcription.

CRISPR/Cas9‑mediated EZH2 knockout suppresses the proliferation and migration of triple‑negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of BC characterized by extensive intratumoral heterogeneity. Compared with other types of BC, TNBC is more prone to invasion and metastasis. The aim of the present study was to determine whether adenovirus-mediated clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system is capable of effectively targeting enhancer of zeste homolog 2 (EZH2) in TNBC cells and lay an experimental basis for the investigation of the CRISPR/Cas9 system as a gene therapy for BC. In the present study, EZH2 was knocked out in MDA-MB-231 cells using the CRISPR/Cas9 gene editing tool to create EZH2-knockout (KO) group (EZH2-KO group). Moreover, the GFP knockout group (control group), and a blank group (Blank group), were employed. The success of vector construction and EZH2-KO were verified by T7 endonuclease I (T7EI) restriction enzyme digestion, mRNA detection and western blotting. Changes in proliferation and migration ability of MDA-MB-231 cells following gene editing were detected by MTT, wound healing, Transwell and in vivo tumor biology assays. As indicated by the results of mRNA and protein detection, the mRNA and protein expression of EZH2 were significantly downregulated in the EZH2-KO group. The difference in EZH2 mRNA and protein between the EZH2-KO and the two control groups was statistically significant. MTT, wound healing and transwell assay suggested that the proliferation and migration ability of MDA-MB-231 cells in the EZH2-KO group were significantly decreased after EZH2 knockout. In vivo, the tumor growth rate in the EZH2-KO group was significantly lower than that in the control groups. In brief, the present study revealed that the biological functions of tumor cells were inhibited after EZH2 knockout in MDA-MB-231 cells. The aforementioned findings suggested that EZH2 can have a key role in the development of TNBC.

Pharmacological inhibition of EZH2 using a covalent inhibitor suppresses human ovarian cancer cell migration and invasion.

Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.

EZH2 as a prognostic-related biomarker in lung adenocarcinoma correlating with cell cycle and immune infiltrates

BackgroundsIt has been observed that high levels of enhancer of zeste homolog 2 (EZH2) expression are associated with unsatisfactory prognoses and can be found in a wide range of malignancies. However, the effects of EZH2 on Lung Adenocarcinoma (LUAD) remain elusive. Through the integration of bioinformatic analyses, the present paper sought to ascertain the effects of EZH2 in LUAD.MethodsThe TIMER and UALCAN databases were applied to analyze mRNA and protein expression data for EZH2 in LUAD. The result of immunohistochemistry was obtained from the HPA database, and the survival curve was drawn according to the library provided by the HPA database. The LinkedOmics database was utilized to investigate the co-expressed genes and signal transduction pathways with EZH2. Up- and down-regulated genes from The Linked Omics database were introduced to the CMap database to predict potential drug targets for LUAD using the CMap database. The association between EZH2 and cancer-infiltrating immunocytes was studied through TIMER and TISIDB. In addition, this paper explores the relationship between EZH2 mRNA expression and NSCLC OS using the Kaplan–Meier plotter database to further validate and complement the research. Furthermore, the correlation between EZH2 expression and EGFR genes, KRAS genes, BRAF genes, and smoking from the Cancer Genome Atlas (TCGA) database is analyzed.ResultsIn contrast to paracancer specimens, the mRNA and protein levels of EZH2 were higher in LUAD tissues. Significantly, high levels of EZH2 were associated with unsatisfactory prognoses in LUAD patients. Additionally, the coexpressed genes of EZH2 were predominantly associated with numerous cell growth-associated pathways, including the cell cycle, DNA replication, RNA transport, and the p53 signaling pathway, according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The results of TCGA database revealed that the expression of EZH2 was lower in normal tissues than in lung cancer tissues (p < 0.05). Smoking was associated with elevated EZH2 expression (p < 0.001). EZH2 was highly expressed in lung cancers with positive KRAS expression, and the correlation was significant in lung adenocarcinoma (r = 0.3129, p < 0.001). CMap was applied to determine the top 15 positively correlated drugs/molecules and the top 15 negatively correlated drugs/molecules. MK-1775, MK-5108, fenbendazole, albendazole, BAY-K8644, evodiamine, purvalanol-a, mycophenolic-acid, PHA-793887, and cyclopamine are potential drugs for patients with lung adenocarcinoma and high EZH2 expression.ConclusionsHighly expressed EZH2 is a predictor of a suboptimal prognosis in LUAD and may serve as a prognostic marker and target gene for LUAD. The underlying cause may be associated with the synergistic effect of KRAS, immune cell infiltration, and metabolic processes.

Sevoflurane Preconditioning Alleviates Posttraumatic Stress Disorder—Induced Apoptosis in the Hippocampus via the EZH2-Regulated Akt/mTOR Axis and Improves Synaptic Plasticity

Posttraumatic stress disorder (PTSD) is a persistent and severe psychological and mental disorder resulting from experiences of serious trauma or stress and is suffered by many individuals. Previous studies have shown that pretreatment with sevoflurane is efficient in reducing the incidence of PTSD. However, we require a more comprehensive understanding of the specific mechanisms by which sevoflurane works. Enhancer of zeste homolog 2 (EZH2) has been reported to be regulated by sevoflurane, and to improve patient cognition. In this study, we aimed to explore the mechanisms of sevoflurane and the role of EZH2 in PTSD cases. We explored the effects of sevoflurane and EPZ-6438 (inhibitor of EZH2) on rat behavior, followed by an investigation of EZH2 mRNA and protein expression. The effects of sevoflurane and EZH2 on neuronal survival were assessed by western blotting and TUNEL staining, while western blotting was used to examine the expression of PSD95 and the AKT/mTOR proteins. Sevoflurane preconditioning restored EZH2 expression and significantly inhibited apoptosis by regulating phosphorylation of the AKT/mTOR pathway. Synaptic plasticity was also significantly improved. These results suggest that pretreatment with sevoflurane could play an important role in PTSD prevention by regulating EZH2 expression.

Clinical Significance of EZH2 in Acute Myeloid Leukemia.

In this paper, we have focused on the investigation of the expression level of the enhancer of zeste homolog 2 (EZH2) gene in bone marrow mononuclear cells of acute myeloid leukemia (AML) patients and analyze the relationship between EZH2 gene expression and EMI. The expression of EZH2mRNA in bone marrow mononuclear cells of 26 patients with incipient AML was detected by qRT-PCR, and the relationship between EZH2mRNA expression and clinical characteristics was analyzed. EZH2 mRNA expression was increased in 26 AML patients. EZH2 gene expression in male patients was significantly higher than that in female patients. The expression of EZH2 in the group with extramedullary infiltration (EMI) was significantly higher than that in the group without EMI. The patients were divided into different groups according to the chromosomal karyotype and prognosis. Statistical analysis showed that the expression level of the medium-risk group was significantly higher than that of the low-risk group, while there was no statistical difference in other groups (P > 0.05). The expression of EZH2 gene in AML patients was closely related to EMI, and the expression of EZH2 in AML cells was closely related to cell migration ability. EZH2 is expected to be one of the indicators of disease recurrence.

Chromatin-Independent Interplay of NFATc1 and EZH2 in Pancreatic Cancer.

Background: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in PDAC progression. Methods: NFATc1 and EZH2 mRNA and protein expression and complex formation were determined in transgenic PDAC models and human PDAC specimens. NFATc1 binding on the Ezh2 gene and the consequences of perturbed NFATc1 expression on Ezh2 transcription were explored by Chromatin Immunoprecipitation (ChIP) and upon transgenic or siRNA-mediated interference with NFATc1 expression, respectively. Integrative analyses of RNA- and ChIP-seq data was performed to explore NFATc1-/EZH2-dependent gene signatures. Results: NFATc1 targets the Ezh2 gene for transcriptional activation and biochemically interacts with the methyltransferase in murine and human PDAC. Surprisingly, our genome-wide binding and expression analyses do not link the protein complex to joint gene regulation. In contrast, our findings provide evidence for chromatin-independent functions of the NFATc1:EZH2 complex and reveal posttranslational EZH2 phosphorylation at serine 21 as a prerequisite for robust complex formation. Conclusion: Our findings disclose a previously unknown NFATc1-EZH2 axis operational in the pancreas and provide mechanistic insights into the conditions fostering NFATc1:EZH2 complex formation in PDAC.

The Role and Significance of Wnt5a in Regulating Epithelial-Mesenchymal Transition in Endometrioid Adenocarcinoma.

PurposeAs a non-classical ligand of Wnt, the abnormal regulation of Wnt5a contributes to the progression of malignant tumors; however, its effects differ depending on tumor type. Here, we evaluated the expression and significance of Wnt5a in endometrioid adenocarcinoma and its relationship with epithelial–mesenchymal transition (EMT)-related proteins.Patients and MethodsImmunohistochemical streptavidin-peroxidase method and reverse transcription polymerase chain reaction (RT-PCR) method were used to analyze the expression and correlation of Wnt5a, and EMT-related protein β-catenin, E-cadherin and enhancer of zeste homolog 2 (EZH2) in endometrial cancer tissues and cell samples of each group.ResultsThe expression of Wnt5a and E-cadherin decreased in the following order, normal endometrium > atypical hyperplasia endometrium > endometrioid adenocarcinoma. In contrast, the expression of β-catenin and EZH2 increased gradually. Moreover, Wnt5a expression was associated with the degree of tissue differentiation, International Federation of Gynecology and Obstetrics (FIGO) stage, and lymph node metastasis (all P<0.05). Wnt5a expression was also negatively correlated with β-catenin and EZH2 expression and positively correlated with E-cadherin expression. RT-PCR results further indicated that E-cadherin mRNA expression was upregulated in a Wnt5a-overexpressing Ishikawa cell line compared to cells transfected with an empty vector or negative control cells (P<0.01). Furthermore, the expression of EZH2 and β-catenin mRNA was downregulated in overexpressing cells compared to empty vector and negative control cells (P<0.01).ConclusionWnt5a may elicit a suppressive effect on endometrioid adenocarcinoma by inhibiting EMT. This study provides a theoretical basis for the pathological diagnosis and targeted therapy of endometrioid adenocarcinoma and extends our understanding of the Wnt5a signaling pathway.

The Tumorigenic Properties of EZH2 are Mediated by MiR-26a in Uveal Melanoma.

Background: The polycomb group protein enhancer of zeste homolog 2 (EZH2) has been found to be highly expressed in various tumors, and microRNA-26a (miR-26a) is often unmodulated in cancers. However, the functions of these two molecules in uveal melanoma (UM) and their relationships have not been reported. Methods: We explored the effects of the miR-26a–EZH2 axis in UM by examining the levels of miR-26a and EZH2. The EZH2 levels in various tumor types and the correlations between EZH2 levels and overall survival and disease-free survival were reanalyzed. The binding of miR-26a to the 3′-untranslated region of EZH2 mRNA was measured using the luciferase reporter assay. The regulation of EZH2 gene expression by miR-26a was also identified, and the effect of elevated EZH2 expression on UM cell function was further examined. Results: miR-26a was downregulated and EZH2 was upregulated in UM cells. Overexpression of miR-26a inhibited cell proliferation, and knockdown of EZH2 suppressed cell growth. EZH2 was a direct target of miR-26a in UM cells. The knockout of EZH2 mimicked the tumor inhibition of miR-26a in UM cells, whereas the reintroduction of EZH2 abolished this effect. In addition, a network of EZH2 and its interacting proteins (UBC, CDK1, HDAC1, SUZ12, EED) was found to participate in miR-26a-mediated tumor progression. Conclusion: The newly identified miR-26a–EZH2 axis may be a potential target for the development of treatment strategies for UM.

Curcumin regulates EZH2/Wnt/β-Catenin pathway in the mandible and femur of ovariectomized osteoporosis rats.

Osteoporosis (OP) behaves in different manners in different parts of the skeleton. This study aims to investigate the effects of curcumin on bone mass of the mandibular and femur from ovariectomized OP rats and to validate whether enhancer of zeste homolog 2 (EZH2)/Wnt/β-Catenin pathway is involved in this process. Curcumin was administered intragastrically into ovariectomized rats for 12 weeks. The bone parameters and the morphology of the trabecular bone of the left mandible and left femur were assessed by micro-computed tomography assay. Morphological changes of the left mandible and left femur were evaluated by hematoxylin and eosin staining. The mRNA levels of EZH2, β-Catenin, and Runx2 in the right mandible and right femur were examined by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to assess EZH2 expression. Both the mandible and femur exhibited OP-like changes in ovariectomized rats, while the mandible bone resorption was less than the femur bone resorption. Curcumin intragastric administration improved bone microstructure and promoted bone formation in the mandible and femur. Curcumin inhibited EZH2 mRNA level and induced that of β-Catenin and Runx2 in the mandible and femur. Collectively, curcumin exerts protective effects against OP, possibly by regulating the EZH2/Wnt/β-Catenin pathway.

Elevated EZH2 in ischemic heart disease epigenetically mediates suppression of NaV1.5 expression

Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

EZH2 is a negative prognostic biomarker associated with immunosuppression in hepatocellular carcinoma.

The enhancer of zeste homolog 2 (EZH2) plays a critical role in different components of anti-tumor immunity. However, the specific role of EZH2 in modulating MHC Class I antigen presentation and T cell infiltration have not been investigated in HCC. This study analyzed the expression and clinical significance of EZH2 in HCC. The EZH2 genetic alterations were identified using cBioPortal. The EZH2 mRNA and protein levels were found to be significantly higher in HCC than in adjacent normal liver tissues in multiple datasets from the GEO and TCGA databases. High expression of EZH2 was significantly correlated with poor overall survival, disease-specific survival, progression-free survival, and relapse-free survival in almost all patients with HCC. The gene set variance analysis (GSVA) showed that the expression of EZH2 is positively correlated with an immunosuppressive microenvironment and negatively correlated with major MHC class I antigen presentation molecules. Gene set enrichment analysis (GSEA) showed that high EZH2 expression is positively associated with the MYC and glycolysis signaling pathway and negatively associated with the interferon-gamma signaling pathway in HCC tissues. These findings demonstrate that EZH2 is a potential prognostic biomarker and therapeutic target in HCC.

EZH2 expression and its role in spermatogonial stem cell self-renewal in goats

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 (H3K27) methyltransferase that plays vital roles in mouse spermatogenesis. However, the expression pattern and role of EZH2 in goat spermatogonial stem cells (SSCs) is unknown. In the present study, we investigated EZH2 expression in the testis of postpubertal goats and its effect on the biological characteristics of goat SSCs. We found that EZH2 mRNA (P < 0.01) and protein (P < 0.05) expression was increased in the testes of postpubertal goats compared to that of prepubertal goats. Moreover, EZH2 was more highly expressed in goat SSCs than in Leydig cells (P < 0.01) and Sertoli cells (P < 0.01) as determined by qPCR, Western blot, and immunofluorescence. Compared to a negative control (NC), cell proliferation (P < 0.01) and viability (P < 0.01) were decreased in SSCs in which EZH2 was knocked down, and the G2/M phase of the cell cycle was blocked (P < 0.01), as determined by Edu staining, CCK-8 assay, and flow cytometry analysis. Additionally, the expression of CASP3, CASP9, and BAX was significantly increased (P < 0.01) while BCL2 expression was decreased (P < 0.01) in EZH2 knockdown SSCs. Notably, the expression of GDNF, a SSCs marker gene, and DAZL, a spermatogenesis-related gene, were significantly decreased (P < 0.01) while GFRA1 expression was significantly up-regulated (P < 0.01) in EZH2 knockdown SSCs. Our data suggest that EZH2 plays a pivotal role in the self-renewal of goat SSCs, and knockdown of EZH2 might impair spermatogenesis in goats.

Effects of miR‑195‑5p on cell proliferation and apoptosis in gestational diabetes mellitus via targeting EZH2.

Gestational diabetes mellitus (GDM) is a type of diabetes mellitus (DM) that occurs during pregnancy. The present study aimed to investigate the roles of microRNA (miR)-195-5p and enhancer of zeste homolog 2 (EZH2) in GDM, and their potential association. Human umbilical vein endothelial cells (HUVECs) were collected from healthy and GDM umbilical cords, and the endothelial properties were detected by flow cytometry. mRNA expression levels of miR-195-5p and EZH2, and EZH2 protein expression levels were detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis, respectively. Cell colony formation and flow cytometry were performed to determine cell proliferation and apoptosis. Furthermore, the target gene of miR-195-5p was predicted and assessed using a dual-luciferase reporter assay. The levels of cell viability, proliferation and apoptosis following the overexpression of miR-195-5p, EZH2 or miR-195-5p + EZH2, were detected using Cell Counting Kit-8, colony formation and flow cytometry assays, respectively. In addition, the mRNA expression levels of miR-195-59 and EZH2, and EZH2 protein expression levels following transfection with overexpression plasmids were detected using RT-qPCR and western blot analysis, respectively. It was identified that high mRNA expression of miR-195-5p, and low EZH2 mRNA and protein expression levels decreased the level of cell proliferation and the high apoptotic rate of GDM-HUVECs. In addition, miR-195-5p was predicted and identified to target EZH2, and miR-195-5p overexpression was identified to inhibit cell proliferation and promote apoptosis. However, it was demonstrated that upregulation of EZH2 could alleviate the inhibition of cell proliferation and the increased apoptotic rate induced by miR-195-5p overexpression. Therefore, the present results suggested that miR-195-5p may inhibit cell viability, proliferation and promote apoptosis by targeting EZH2 in GDM-induced HUVECs.

LncRNA-ANCR down-regulation suppresses invasion and migration of colorectal cancer cells by regulating EZH2 expression.

Our study aimed to explore the effects of long noncoding RNA (lncRNA)-ANCR on the invasion and migration of colorectal cancer (CRC) cells by regulating enhancer of zeste homolog 2 (EZH2) expression. CRC tissues and adjacent normal tissues were collected and CRC SW620 cells line and normal human intestinal epithelial cells (HIECs) were incubated. CRC SW620 cells line was transfected with ANCR-siRNA. The expressions of ANCR and EZH2 mRNA were measured by real-time quantitative polymerase chain reaction (RT-qPCR). EZH2 and trimethylation of H3K27 (H3K27me3) protein expressions were detected using Western blotting. The relationship between ANCR and EZH2 was determined through RNA pull-down and co-immunoprecipitation (co-IP) assays. Cell invasion and migration were determined by Trans-well and cell scratch assays. ANCR, EZH2 and H3K27me3 expressions were up-regulated in CRC tissues and SW620 cells (all P < 0.05). After transfected with ANCR-siRNA, SW620 cells showed decreased ANCR expression and EZH2 mRNA and protein expressions (all P < 0.05). According to the results of RNA pull-down and co-IP assays, ANCR could specifically bind to EZH2. The results of Trans-well and cell scratch tests showed that when ANCR expression was decreased, the invasion and migration abilities of SW620 cells significantly declined (both P < 0.05). In conclusion, these results suggest that lncRNA-ANCR could influence the invasion and migration of CRC cells by specifically binding to EZH2.

MicroRNA-130-5p promotes invasion as well as migration of lung adenocarcinoma cells by targeting the EZH2 signaling pathway.

This study was designed to investigate whether microRNA-130-5p could promote the progression and metastasis of lung cancer by targeting enhancer of zeste homolog 2 (EZH2). The level of microRNA-130-5p was examined in the tumor tissues and paracancerous tissues of lung adenocarcinoma patients. Meanwhile, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the level of microRNA-130-5p in different lung adenocarcinoma cell lines, which was found markedly decreased in two lung cancer cell lines. Moreover, microRNA-130-5p mimic and inhibitor were transfected in cells to achieve microRNA-130-5p overexpression and knockdown model to observe its effect on cell biological function. Cell Counting Kit-8 (CCK-8) assay was performed to detect cell viability, while transwell assay was used to detect cell invasion as well as migration. Subsequently, EZH2 was predicted as the target gene of microRNA-130-5p using the Bioscan prediction site Targetscan, which was confirmed by Luciferase reporter gene assay. Besides, the level of EZH2 was detected by qRT-PCR and Western blot after overexpression and knockdown of microRNA-130-5p. Finally, the combination of si-EZH2 and inhibitor was used to further verify whether microRNA-130-5p could promote cell metastasis and invasion of lung cancer by targeting EZH2. MicroRNA-130-5p expression in tumor tissues of patients with lung adenocarcinoma was markedly lower than that in normal tissues. Besides, microRNA-130-5p expression in tumor tissues with large diameter was lower than that with a small diameter. In addition, microRNA-130-5p was also lowly expressed in lung cancer cell lines. High expression of microRNA-130-5p reduced the cell viability and inhibited cancer cell metastasis and invasion. At the same time, microRNA-130-5p knockdown enhanced the activity of lung cancer cells and promoted cancer cell invasion as well as migraindicated confirmed that microRNA-130-5p could bind to EZH2. Additionally, the overexpression or knockdown of microRNA-130-5p could decrease or increase the levels of EZH2 mRNA, respectively. Finally, EZH2 silencing reduced lung cancer cell activity and inhibited cancer cells invasion as well as migration, which could be reversed by the microRNA-130-5p inhibitor. The reduced microRNA-130-5p expression in lung cancer tissues and cells promoted metastasis and invasion of this tumor by targeting EZH2.

Oncogenic enhancer of zeste homolog 2 is an actionable target in patients with non-small cell lung cancer.

BackgroundThe aims of this study were to investigate the link between enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDAC) in preclinical studies and in human lung cancer tissue microarrays.MethodsEnhancer of zeste homolog 2 and HDAC1 mRNA expression in two lung adenocarcinoma (LUAD) datasets (MDACC and TCGA) were correlated with patient outcomes. We evaluated the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor, suberoylanilide hydroxamic acid (SAHA). The response to SAHA was assessed at baseline and after alteration of EZH2 or HDAC mRNA expression in LUAD cell lines.ResultsDirect correlation was found between EZH2 and HDAC1 expression (P < 0.0001). When EZH2 expression was knocked down‐ or upregulated, there was a corresponding decrease or increase in expression of HDAC expression, respectively. Cell lines with high EZH2 expression responded to SAHA treatment with a mean inhibition rate of 73.1% compared to 43.2% in cell lines with low EZH2 expression (P < 0.0001). This correlation was confirmed in non‐small cell lung cancer (NSCLC) specimens from MDACC (Spearman's correlation r = 0.416; P < 0.0001) and TCGA datasets (r = 0.221; P < 0.0001). Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of both datasets had the lowest survival compared to the patients with low expression of either or both markers.ConclusionOur findings show that overexpression of EZH2 is a negative prognostic indicator. Increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting NSCLC patients for treatment with HDAC inhibitors.

Abstract 4728: IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma

Abstract Background: The molecular mechanisms governing anti-PD-1 resistance in melanoma are not fully understood. Suboptimal transcriptional and molecular response to interferon-gamma (IFNγ) is commonly identified in tumors with intrinsic or acquired resistance to anti-PD-1. Epigenetic regulators have been shown to reprogram IFNγ responses in some cases, though the mechanism is not clear. The ability of melanoma cells to express MHC-II is a positive clinical predictor to anti-PD-1 therapy, and MHC-II is induced only on cells with robust IFNγ responses. Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator with both methyltransferase-dependent and -independent function, can suppress IFNγ target genes and is frequently overexpressed in melanoma. However, the mechanisms whereby EZH2 interacts with the IFNγ pathway and its role in melanoma-specific MHC-II expression is not known. Methods: HLA-DR (MHC-II)-proficient (A375, SKMEL28 and SKMEL5) and -deficient (CHL-1 and MEWO) melanoma cell lines were stimulated with IFNγ for up to 48h, and the modulation of EZH2 protein and function were studied temporally. To test the role of EZH2 in MHC-II (HLA-DR) expression in IFNγ responses, genetic (siRNA) and chemical (GSK343) EZH2 inhibition was utilized. Results: EZH2 was highly expressed in all cell lines tested, regardless of HLA-DR-proficiency. In 2 out of 3 HLA-DR-inducible melanoma cell lines, EZH2 protein expression was downregulated (4-24 hrs) in response to IFNγ stimulation, without effects on EZH2 mRNA expression or downstream tri-methylated H3K27 expression. Blocking proteosomal degradation with MG-132 reversed the IFNγ-induced decrease in EZH2 expression in HLA-DR-proficient cell lines. After siRNA knockdown of EZH2, SKMEL28 cells increased constitutive HLA-DR expression, while A375 cells became more sensitive to IFNγ-induced HLA-DR upregulation. In addition, IFNγ-induced HLA-DR was increased in A375 cells with EZH2 inhibition using GSK343. Conclusion: This work demonstrates a potential role for EZH2 in suppressing IFNγ responses, including the induction of MHC-II, in melanoma cells. EZH2 is subject to proteosomal degradation in MHC-II-inducible cell lines, which, in part, contributes to the expression of MHC-II. Citation Format: Jamaal L. James, Susan R. Opalenik, Abigail Toren, Rebecca S. Cook, Justin M. Balko. IFNγ signaling drives EZH2 degradation to induce MHC-II expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4728.

Effects of enhancer of Zeste homolog 2 inhibitor GSK126 on the cell proliferation and apoptosis of acute leukemia and lymphoma cells in vitro

Objective To investigate the effects of a novel enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 in vitro on the cell proliferation and apoptosis in acute leukemia and lymphoma cells. Methods CCK-8 assay was used to measure the effects of GSK126 with different concentrations and time on the cell proliferation in human T-cell acute lymphoblastic leukemia (T-ALL) CEM cell line, acute monocytic leukemia U937 cell line and Burkitt lymphoma Raji cell line. Annexin V/PI double staining method was used to determine the effects of GSK126 on the cell apoptosis. The effect of GSK126 on the mRNA levels of EZH2, bcl-2 and bcl-xL were measured by using quantitative polymerase chain reaction (qPCR). Results After the function for 24 h, 48 h and 72 h, compared to the negative control group (0 μmol/L), 5, 10, 15, 20, 25 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in CEM cells; 5, 10, 15, 20 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in U937 cells; 5, 10, 15, 20, 25, 30 μmol/L GSK126 treatment showed a significant time-dependent suppression in Raji cells (all P < 0.05). The 48 h 50% inhibitory concentration (IC50) of GSK126 on CEM, U937, Raji cells was (13.46±0.83), (11.65±1.02), (15.00±0.19) μmol/L, respectively. GSK126 treatment with the doses of 8, 12 and 16 μmol/L promoted the apoptosis of CEM and U937 cells compared with the negative control group (0 μmol/L), and there were statistical differences in the apoptosis rate (F = 167.995, P < 0.01; F = 158.400, P < 0.01). In Raji cells, only 16 μmol/L GSK126 treatment had a higher cell apoptosis rate compared with the control group (t = 47.998, P < 0.05). Furthermore, 8, 12 and 16 μmol/L GSK126 treatment suppressed the expressions of EZH2 mRNA in CEM, U937 and Raji cells compared with the control group (F = 82.035, P < 0.01; F = 252.712, P < 0.01; F = 690.536, P < 0.01), and the expressions of bcl-2 and bcl-xL mRNA (bcl-2: F = 1 900.525, P < 0.01; F = 431.324, P < 0.01; F = 216.184, P < 0.01; bcl-xL: F = 256.751, P < 0.01; F = 147.019, P < 0.01; F = 209.325, P < 0.01). Conclusion EZH2 plays an important role in the occurrence and development of acute leukemia and lymphoma. GSK126 as a high selective inhibitor of EZH2 might be a potential new drug in treatment of hematological malignancies. Key words: Hematologic neoplasms; Cell proliferation; Apoptosis; Enhancer of Zeste homolog 2; GSK126

&lt;p&gt;LncRNA SNHG6 promotes the migration, invasion, and epithelial-mesenchymal transition of colorectal cancer cells by miR-26a/EZH2 axis&lt;/p&gt;

Objective: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Small nucleolar RNA host gene 6 (SNHG6) was reported to function as an oncogene in a number of cancers. Here, we aimed to further explore the roles and molecular mechanism of SNHG6 in CRC metastasis.Methods: The expression levels of SNHG6, miR-26a, and enhancer of zeste homolog 2 (EZH2) mRNA were assessed by quantification real-time PCR in CRC tissues and cell lines. Western blot analysis was performed to determine the levels of E-cadherin, Snail, Vimentin, N-cadherin, and EZH2. Cell migration and invasion capacities were detected by transwell assay. Dual-luciferase reporter assay or RNA Immunoprecipitation assay was employed to verify the interaction between SNHG6 and miR-26a, or EZH2 and miR-26a.Results: Our data indicated that SNHG6 and EZH2 mRNA were upregulated, and miR-26a was downregulated in CRC tissues and cell lines. SNHG6 knockdown suppressed the migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells. Moreover, SNHG6 binded to miR-26a and repressed miR-26a expression. EZH2 was a direct target of miR-26a, and it was regulated by SNHG6/miR-26a. MiR-26a inhibitor undermined the effect of SNHG6 knockdown on cell migration, invasion, and EMT. Additionally, EZH2 antagonized the effect of miR-26a on cell migration, invasion, and EMT in CRC cells.Conclusion: SNHG6 knockdown suppressed cell migration, invasion, and EMT at least partly by sponging miR-26a and regulating EZH2 expression in CRC cells, providing a strategy for blocking CRC metastasis.