Enhancer Of Zeste Homolog 2 Knockdown Research Articles

Dysregulated lipid metabolism blunts the sensitivity of cancer cells to EZH2 inhibitor.

SummaryBackgroundSensitivity has been a key issue for Enhancer of zeste homolog 2 (EZH2) inhibitors in cancer therapy. The EZH2 inhibitor EPZ-6438 was first approved by the US Food and Drug Administration (FDA) in 2020. However, its inadequate anti-cancer activity in solid tumors limits its clinical application. In this study, we utilized the multiple cancer cell lines, which are less sensitive to the EZH2 inhibitor GSK126, combining animal model and clinical data to investigate the underlying mechanism.MethodsIncuCyte S3 was used to explore the difference in the responsiveness of hematological tumor cells and solid tumor cells to GSK126. Transcriptome and metabolome of B16F10 cells after GSK126 treatment were analyzed and the distinct changes in the metabolic profile were revealed. Real-time quantitative PCR and western blot experiments were used to further verify the multi-omics data. ChIP-qPCR was performed to detected H3K27me3 enrichment of target genes. Finally, the anti-tumor effects of combining GSK126 and lipid metabolism drugs were observed with IncuCyte S3 platform, CCK-8 and animal model respectively.FindingsWe found that although the proliferative phenotype did not show strong difference upon treatment with GSK126, the transcriptome and metabolome changed profoundly. GSK126 treatment led to broad shifts in glucose, amino acid, and lipid metabolism. Lipid synthesis was strengthened manifested by the increasing abundance of unsaturated fatty acids. SCD1 and ELOVL2 were regulated by H3K27me3 at gene regulatory region, and upregulated by EZH2 knockdown and inhibitors. SCD1 knockdown increased cellular sensitivity to GSK126. Based on the findings above, the application of the combination with SCD1 inhibitor significantly attenuated the proliferation of cancer and increased the sensitivity to GSK126 by suppressing desaturation of fatty acids.InterpretationDysregulated lipid metabolism can blunt the sensitivity of cancer cells to GSK126. These characteristics shed light on the novel combination therapy strategies to combat tumor resistance.FundingNational Natural Science Foundation of China (No. 81672091, No.91749107 and No. 81972966).

Silencing of MBD2 and EZH2 inhibits the proliferation of colorectal carcinoma cells by rescuing the expression of SFRP.

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

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.

Development and Characterization of the Novel Orally Bioavailable EZH1/2 Dual Inhibitor DS-3201

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27) and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 function as catalytic subunits of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Many reports suggest that inhibiting PRC2 activity through EZH2 knockdown gives rise to anti-cancer effect against hematological and solid cancers.To date, some EZH2 selective inhibitors have demonstrated anti-cancer potential in preclinical and clinical studies. In spite of this promise, there is concern that the efficacy anticipated with EZH2 selective inhibitors may be compromised due to compensation by EZH1 activity.Herein, we describe the novel, orally bioavailable EZH1/2 dual inhibitor DS-3201. This compound strongly and selectively inhibited the methyltransferase activity of both EZH1 and EZH2 with an IC50 value of 10 nM and 6.0 nM, respectively in cell-free enzymatic assays. This compound also suppressed trimethylation of H3K27 in cells (IC50: 0.55 nM) more potently than EZH2 selective inhibitors.In order to identify the anti-cancer spectrum of DS-3201, we tested its in vitro growth inhibition activity against a panel of cancer cell lines including a variety of hematological cancers. Among the panel, hematological cell lines showed particular sensitivity to DS-3201, with GI50 values less than 100 nM, including 1) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines harboring fusion genes, such as MLL-AF9, MLL-AF4, or AML1-ETO, 2) diffuse large B-cell lymphoma (DLBCL) cell lines having EZH2 gain-of-function (GOF) mutations, 3) most of the peripheral T-cell lymphoma and multiple myeloma cell lines tested. Our data thus shows this compound has broader anti-cancer activity than EZH2 selective inhibitors, especially in hematology cancers. We also evaluated the in vivo anti-tumor efficacy of DS-3201 against KARPAS-422, a DLBCL tumor xenograft with an EZH2 GOF mutation. A 100 mg/kg once daily oral administration of DS-3201 caused nearly complete tumor regression without any body weight loss and 25 mg/kg administration also showed clear tumor growth delay.Based on these results, we initiated clinical studies of DS-3201 for patients with lymphoma (Clinical trial information: NCT02732275) and for those with AML and ALL (NCT03110354). DisclosuresHonma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Kanno:Asubio Pharma Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Hirasawa:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Takizawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Yasumatsu:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Horiuchi:Daiichi Sankyo Co., Ltd.: Employment. Nakao:Daiichi Sankyo Co., Ltd.: Employment. Suzuki:Daiichi Sankyo Co., Ltd.: Employment. Yamasaki:Daiichi Sankyo Co., Ltd.: Employment. Nakajima:Daiichi Sankyo Co., Ltd.: Employment. Hayakawa:Daiichi Sankyo Co., Ltd.: Employment. Yamazaki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Fujiwara:Daiichi Sankyo Co., Ltd.: Employment.

EZH2 regulates the malignancy of human glioblastoma cells via modulation of Twist mRNA stability

Glioblastoma multiforme (GBM) is a lethal primary brain tumor with poor survival lifespan and dismal outcome. However, the effects and mechanisms of epigenetic factors on the development of GBM were still not well illustrated. We found that expression of enhancer of zeste homolog 2 (EZH2), which can catalyze histone H3K27me3 to modulate gene expression, was increased in GBM cells. Knockdown of EZH2 can suppress proliferation and migration, while increase temozolomide (TMZ) sensitivity, of GBM cells. Further, knockdown of EZH2 or its specific inhibitor GSK126 can decrease expression of Twist, while over expression of Twist can reverse si-EZH2-suppressed malignancy of GBM cells. Mechanistically, EZH2 can positively regulate mRNA stability of Twist1 mRNA. Further, miR-206, which can bind with 3′UTR of Twist1 mRNA, was involved in EZH2-regulated mRNA stability of Twist1. Collectively, our data suggest that EZH2 might be a potential target for GBM treatment. Further, miR-206/Twist axis is involved in EZH2-regulated malignancy of GBM cells.

Role of enhancer of zeste homolog 2 in osteoclast formation and periodontitis development by downregulating microRNA-101-regulated VCAM-1.

The enhancer of zeste homolog 2 (EZH2) represents a potential target for periodontitis treatment; however, its role in the development of periodontitis remains unclear. The current study aimed to elucidate the role of EZH2 in osteoclasts (OCs) growth as well as the mechanism underpinning the related process. The potential interaction among EZH2, microRNA-101 (miR-101), and vascular cell adhesion molecule 1 (VCAM-1) was evaluated using chromatin immunoprecipitation and dual-luciferase reporter gene assay. The expressions of EZH2 and miR-101 in OCs were examined by Western blot analysis and reverse transcription squantitativepolymerase chain reaction. Loss- and gain-function assays were then performed to determine the role of EZH2/miR-101/VCAM-1 in periodontitis and OCs proliferation, followed by OC growth and proliferation detected using tartrate resistant acid phosphatase (TRAP) and 5-ethynyl-2'-deoxyuridine staining. Enzyme-linked immunoassay was conducted to determine the expression of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α). A periodontitis rat model was established to investigate the effect of EZH2 and VCAM-1 in vivo. EZH2 was overexpressed, while miR-101 was downregulated in the OCs of periodontitis. Silencing of EZH2, VCAM-1 repression, or miR-101 elevation suppressed the growth and proliferation of OC while acting to encumber the release of IL-1β and TNF-α. EZH2 negatively targeted miR-101, while miR-101 negatively targeted VCAM-1. Moreover, silencing of EZH2 or VCAM-1 was observed to attenuate periodontitis which was evidenced by an increase in BMD, BV/TV, and BS/BV as well as reduction in TRAP and cathepsin K in vivo. Taken together, the key findings of the current study demonstrate that EZH2 knockdown inhibited OC formation by elevating the expression of miR-101 via suppression of VCAM-1, ultimately attenuating periodontitis.

Implication of EZH2 in the Pro-Proliferative and Apoptosis-Resistant Phenotype of Pulmonary Artery Smooth Muscle Cells in PAH: A Transcriptomic and Proteomic Approach.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling.

LINC10536 Attenuates Lung Adenocarcinoma Proliferation and Metastasis

Background: LUAD is a devastating disease with poor patient prognosis. Long non-coding RNAs (lncRNAs) have been implicated to play a critical role in LUAD progression. Methods: Differentially expressed and methylated tumor suppressor lncRNAs were found using TCGA, GSE81089) and E-MTAB-6957 datasets. The potential biological functions of LINC10536 were examined by loss- and gain-of-function models. Potential downstream pathway was validated by quantitative real-time polymerase chain reaction (RT-qPCR). RNA immunoprecipitation (RIP) arrays were employed to validate the binding between LINC10536 and enhancer of zeste homologue 2 (EZH2). Chromatin immunoprecipitation (CHIP) assays were used to demonstrate CDKN1B and E-cadherin as direct targets of EZH2. Results: The landscape of methylation-mediated tumor suppressor lncRNAs for LUAD were identified in this study. Overexpression of LINC10536 suppressed cell proliferation, cell cycle, invasion, migration and EMT of LUAD cells, whereas knockdown of LINC10536 promoted proliferative and metastatic potential of lung cancer cells in vitro and in vivo. As a nuclear lncRNA LINC10536 knockdown enhanced the occupancy capacity of EZH2 and H3K27me3 at the promoter region of CDKN1B and E-cadherin utilizing CHIP-qPCR. EZH2 knockdown partially reverses the malignant phenotype mediated by shLINC10536 in vitro and in vivo. Moreover, DNA methyltransferase inhibitor 5-azacytosine significantly facilitated LINC10536 expression and its biological functions. Interpretation: Our finding suggests LINC10536 might be a promising target for cancer diagnosis and therapy in clinical practice. Fundings: This project is partially supported by the National Natural Science Foundation of China (81772474, 82072563 to LC and 81803023 to YX). Declaration of Interest: The authors declare that they have no conflict of interest.

EZH2 inhibition decreases neuroblastoma proliferation and in vivo tumor growth.

Investigation of the mechanisms responsible for aggressive neuroblastoma and its poor prognosis is critical to identify novel therapeutic targets and improve survival. Enhancer of Zeste Homolog 2 (EZH2) is known to play a key role in supporting the malignant phenotype in several cancer types and knockdown of EZH2 has been shown to decrease tumorigenesis in neuroblastoma cells. We hypothesized that the EZH2 inhibitor, GSK343, would affect cell proliferation and viability in human neuroblastoma. We utilized four long-term passage neuroblastoma cell lines and two patient-derived xenolines (PDX) to investigate the effects of the EZH2 inhibitor, GSK343, on viability, motility, stemness and in vivo tumor growth. Immunoblotting confirmed target knockdown. Treatment with GSK343 led to significantly decreased neuroblastoma cell viability, migration and invasion, and stemness. GSK343 treatment of mice bearing SK-N-BE(2) neuroblastoma tumors resulted in a significant decrease in tumor growth compared to vehicle-treated animals. GSK343 decreased viability, and motility in long-term passage neuroblastoma cell lines and decreased stemness in neuroblastoma PDX cells. These data demonstrate that further investigation into the mechanisms responsible for the anti-tumor effects seen with EZH2 inhibitors in neuroblastoma cells is warranted.

Overexpression of EZH2/NSD2 Histone Methyltransferase Axis Predicts Poor Prognosis and Accelerates Tumor Progression in Triple-Negative Breast Cancer.

Two histone methyltransferases, enhancer of zeste homolog 2 (EZH2) and nuclear SET domain-containing 2 (NSD2), are aberrantly expressed in several types of human cancers. However, the regulatory relationship between EZH2 and NSD2 and their prognostic values in breast cancer (BC) have not been fully elucidated. In this study, we demonstrated that EZH2 and NSD2 were overexpressed in BC compared with benign lesions and normal tissues using tissue microarray, immunohistochemistry, and bioinformatic databases. Both EZH2 and NSD2 expression were associated with pathological grade of tumor and lymph node metastasis. A comprehensive survival analysis using Kaplan-Meier Plotter database indicated that EZH2 expression was negatively correlated with relapse-free survival (RFS), overall survival (OS), distant metastasis-free survival (DMFS), and postprogression survival (PPS) in 3951 BC patients, and NSD2 expression was negatively correlated with RFS and DMFS. Notably, EZH2 and NSD2 expression were coordinately higher in triple-negative breast cancer (TNBC) than that in other subtypes. Stable knockdown of EZH2 using lentiviral shRNA vector significantly reduced the proliferation, migration and invasion abilities of TNBC cell line MDA-MB-231 and MDA-MB-468, and downregulated NSD2 expression as well as the levels of H3K27me3 and H3K36me2, two histone methylation markers catalyzed by EZH2 and NSD2, respectively. By contrast, overexpression of EZH2 using adenovirus vector displayed an inverse phenotype. Furthermore, knockdown of NSD2 in EZH2-overexpressing cells could dramatically attenuate EZH2-mediated oncogenic effects. Bioinformatic analysis further revealed the function and pathway enrichments of co-expressed genes and interactive genes of EZH2/NSD2 axis, suggesting that EZH2/NSD2 axis was associated with cell division, mitotic nuclear division and transition of mitotic cell cycle in TNBC. Taken together, EZH2/NSD2 axis may act as a predictive marker for poor prognosis and accelerate the progression of TNBC.

Targeting EZH2 Ameliorates the LPS-Inhibited PDLSC Osteogenesis via Wnt/β-Catenin Pathway

As a histone methyltransferase, enhancer of zeste homolog 2 (EZH2), suppresses osteoblast maturation and is involved in inflammation. However, the role of EZH2 in human periodontal ligament stem cells (PDLSCs) under inflammation still needs to be further investigated. This study aimed to identify the underlying mechanisms and explore the function of EZH2 in PDLSC osteogenesis under inflammation. PDLSCs were treated with sh-EZH2, DZNep or DKK1 under inflammation. The alkaline phosphatase (ALP) activity, alizarin red staining, and osteogenesis-related protein levels were analyzed. Lipopolysaccharide (LPS)-induced inflammation restrained osteogenic differentiation. Under inflammation, the upregulation of EZH2 suppressed the expression of osteogenic markers, including osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2, the activity of ALP, and the accumulation of mineralization through the Wnt/β-catenin pathway. EZH2 knockdown inhibited the levels of proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α. These results suggested that LPS-induced overexpression of EZH2 suppressed PDLSC osteogenesis under inflammatory conditions through the Wnt/β-catenin pathway. These findings give new insights into the physiological differentiation and pathological inflammation of PDLSC osteogenesis, and provide an underlying therapeutic target for periodontitis.

EZH2 activates CHK1 signaling to promote ovarian cancer chemoresistance by maintaining the properties of cancer stem cells.

Background: Ovarian cancer is a fatal malignant gynecological tumor. Ovarian cancer stem cells (OCSCs) contribute to resistance to chemotherapy. The polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a key role in maintaining CSCs. Here, we aimed to investigate the specific mechanism by which EZH2 regulates CSCs to result in chemoresistance and poor prognosis of ovarian cancer.Methods: We used a nude mouse model to obtain a cell line enriched for OCSCs, named SK-3rd cells. The CRISPR and Cas9 endonuclease system was used to establish an EZH2-knockout SK-3rd ovarian cancer cell line. High-throughput PCR array and bioinformatics methods were used to screen the EZH2 target involved in CSC stemness. A luciferase reporter assay and chromatin immunoprecipitation assay were performed to identify activation of CHK1 by EZH2. We evaluated associations between EZH2/CHK1 expression and the chemoresistance and prognosis of ovarian cancer patients.Results: EZH2 plays a critical role in maintaining ovarian CSC stemness and chemo-resistance. CHK1 is an EZH2 target involved in CSC stemness. Knockdown of EZH2 in ovarian CSCs decreased CHK1 expression, while CHK1 overexpression was sufficient to reverse the inhibitory effect on spheroid formation and chemoresistance caused by repression of EZH2. In addition, EZH2 was also shown to play a unique role in activating rather than repressing CHK1 signaling through binding to the CHK1 promoter in epithelial ovarian cancer cells. Finally, in clinical samples, ovarian cancer patients with high levels of EZH2 and CHK1 not only were more resistant to platinum but also had a poorer prognosis.Conclusions: Our data revealed a previously unidentified functional and mechanistic link between EZH2 levels, CHK1 signaling activation, and ovarian CSCs and provided strong evidence that EZH2 promotes ovarian cancer chemoresistance and recurrence.

EZH2 is involved in psoriasis progression by impairing miR-125a-5p inhibition of SFMBT1 and leading to inhibition of the TGFβ/SMAD pathway.

Aims:In this study, we aimed to decipher the impact of enhancer of zeste homolog 2 (EZH2) in psoriasis as well as the underlying mechanism.Methods:A mouse model of psoriasis was developed by means of imiquimod induction, with the expression of EZH2, microRNA-125a-5p (miR-125a-5p), and SFMBT1 determined. The role of EZH2, miR-125a-5p, and SFMBT1 in malignant phenotypes of HaCaT cells and the development of psoriasis in vivo was subsequently investigated through gain- and loss-of-function experiments. Chromatin immunoprecipitation assay and dual-luciferase reporter assay were conducted to explore the relationship between EZH2 or SFMBT1 and miR-125a-5p. Finally, the effects of EZH2 and miR-125a-5p on the transforming growth factor β (TGFβ)/SMAD pathway were analyzed.Results:Overexpressed SFMBT1 and EZH2 was detected while miR-125a-5p were downregulated in psoriasis tissues and human keratinocyte (HaCaT) cells. EZH2 increased the levels of IL-17A-induced cytokines and promoted the malignant phenotypes of HaCaT cells. Functionally, EZH2 reduced miR-125a-5p expression while miR-125a-5p targeted SFMBT1 to activate the TGFβ/SMAD pathway in vitro. Knockdown of EZH2 or up-regulation of miR-125a-5p inhibited cell proliferation and the levels of IL-17A-induced cytokines, but increased the expression of TGFβ1 and the extent of smad2 and smad3 phosphorylation in HaCaT cells. Notably, EZH2 contributed to the development of psoriasis in vivo by inhibiting the TGFβ/SMAD pathway via impairment of miR-125a-5p-mediated SFMBT1 inhibition.Conclusion:Taken together, the results of the current study highlight the ability of EZH2 to potentially inactivate the TGFβ/SMAD pathway via upregulation of miR-125a-5p-dependent SFMBT1during the progression of psoriatic lesions.

Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts.

BackgroundMicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown.MethodsWe harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different in vitro conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo.ResultsWe found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3′-untranslated region (3′UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model.ConclusionsOur data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.

Enhancer of Zeste Homolog 2 (EZH2) Mediates Glucolipotoxicity-Induced Apoptosis in β-Cells.

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced β-cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the β-cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. β-Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NFκB pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

MBD2 and EZH2 regulate the expression of SFRP1 without affecting its methylation status in a colorectal cancer cell line.

Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.

Epigenetic silencing of ZIC4 contributes to cancer progression in hepatocellular carcinoma

Inactivation of tumor suppressor gene played critical roles in the development and progression of human hepatocellular carcinoma (HCC). Zic family member 4 (ZIC4) is transcription factor and plays an important role in the developmental process. However, the expression and biological role of ZIC4 in HCC is poorly understood. Here, bioinformatics analysis based on The Cancer Genome Atlas (TCGA) database revealed an aberrant hypermethylation of ZIC4 in HCC. ZIC4 is frequently hypermethylated in promoter region and down expressed in HCC cells and tissues. Functionally, ZIC4 inhibition facilitated the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Conversely, ZIC4 overexpression reduced proliferation and invasiveness of HCC cells. In addition, ZIC4 inhibition rescued the antitumor effect induced by enhancer of zeste homolog 2 (EZH2) knockdown or EZH2 inhibitor. Mechanistically, EZH2 knockdown or EZH2 inhibitor reduced the enrichment of EZH2 and H3K27me3 in ZIC4 promoter region and leading to the upregulation of ZIC4. Altogether, these data indicate that epigenetic silencing of ZIC4 by EZH2 mediated H3K27me3 is an important mechanism in HCC and provide a new therapeutic target for the treatment of hepatocellular carcinoma disease.

Determination of long non‑coding RNAs associated with EZH2 in neuroblastoma by RIP‑seq, RNA‑seq and ChIP‑seq

Neuroblastoma (NB) is the most common type of extracranial solid tumor found in children. Despite several treatment options, patients with advanced stage disease have a poor prognosis. Previous studies have reported that enhancer of zeste homolog 2 (EZH2) and long non-coding RNAs (lncRNAs) have abnormal expression levels in NB and participate in tumorigenesis and NB development. However, the association between EZH2 and lncRNAs remain unclear. In the present study, RNA immunoprecipitation-sequencing (RIP-seq) was used to analyze the lncRNAs binding to EZH2. Following EZH2 knockdown via short hairpin RNA, RNA-seq was performed in shEZH2 and control groups in SH-SY5Y cells. Chromatin IP (ChIP)-seq was used to determine the genes that may be regulated by EZH2. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to identify the signaling pathways involved in NB. The results from RIP-seq identified 94 lncRNAs, including SNHG7, SNHG22, KTN-AS1 and Linc00843. Furthermore, results from RNA-seq demonstrated that, following EZH2 knockdown, 448 genes were up- and 571 genes were downregulated, with 32 lncRNAs up- and 35 downregulated and differentially expressed compared with control groups. Certain lncRNAs, including MALAT1, H19, Linc01021 and SNHG5, were differentially expressed in EZH2-knockdown group compared with the control group. ChIP-seq identified EZH2 located in the promoter region of 138 lncRNAs including CASC16, CASC15, LINC00694 and TBX5-AS1. In summary, the present study demonstrated that certain lncRNAs directly bound EZH2 and regulated EZH2 expression levels. A number of these lncRNAs that are associated with EZH2 may participate in NB tumorigenesis.

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.

MiR‑144‑3p inhibits tumor cell growth and invasion in oral squamous cell carcinoma through thedownregulation of the oncogenic gene, EZH2.

Accumulating evidence demonstrates that microRNAs (miRNAs or miRs) play important roles in the development and progression of human malignancies, including oral squamous cell carcinoma (OSCC); however, the unique roles of miRNAs are not yet fully understood in OSCC. The present study aimed to identify novel miRNAs associated with OSCC and to elucidate their functions. Based on a micro-array analysis, miR-144-3p was found to be one of the most significantly downregulated miRNAs in OSCC tissues. Its low expression was closely associated with tumor size, differentiation and lymph node metastasis. Functionally, miR-144-3p overexpression suppressed proliferation, promoted apoptosis, and suppressed the invasion and migration of OSCC cells. In addition, enhancer of zeste homolog 2 (EZH2), a well-known oncogene, was proven to be a direct target of miR-144-3p, and its protein expression was negatively regulated by miR-144-3p. Moreover, EZH2 expression was increased, and inversely correlated with the miR-144-3p level in OSCC tissues. Notably, EZH2 knockdown inhibited cell proliferation, promoted cell apoptosis, and suppressed the invasion and migration of OSCC cells, whereas EZH2 overexpression partially reversed the anticancer effects mediated by miR-144-3p overexpression. On the whole, the findings of the present study suggest that miR-144-3p functions as a tumor suppressor by targeting the EZH2 oncogene, and may thus be considered as a potential diagnostic and therapeutic target for OSCC.