Role of Histone Methyltransferase in Fibrosis and Traditional Chinese Medicine Treatment： A Review / 中国实验方剂学杂志

Fibrosis， a tumor-like lesion between benign tissue and malignant tumor， mostly occurs in the liver， kidney， heart， lung， bone marrow and other organs and tissues. It can affect almost every organ and eventually induce multiple organ failure and cancers， seriously endangering human life. It will be of great importance to prevent cancer if the disease can be opportunely blocked in the fibrotic stage. The pathogenesis of fibrosis is still not completely clear. It is of great clinical significance to study the occurrence， development， and mechanism of fibrosis as well as to screen new therapeutic targets. Enhancer of zeste homolog 2 （EZH2） is mainly located in the nucleus and involved in the formation of the polycomb repressive complex 2. EZH2 is a methyltransferase which makes the lysine on position 27 of histone H3 （H3K27me3） undergo trimethyl modification induces gene silencing through classical or nonclassical actions， so as to inhibit or activate transcription. EZH2 plays a critical role in cell growth， proliferation， differentiation， and apoptosis， which is regulated by different targets and signaling pathways. EZH2 regulates the transformation of myofibroblasts and participates in the fibrosis of multiple organs. Recent studies have shown that EZH2 plays a role in fibrosis-related pathophysiological processes such as epithelial-mesenchymal transition， oxidative stress， and inflammation. EZH2 as the target of fibrosis， EZH2 inhibitors， and EZH2-related traditional Chinese medicine （TCM） formula and active compounds have gradually become hot research directions. EZH2 may be a powerful target for organ fibrosis. Exploring the structure， function， and distribution of EZH2， the role of EZH2 in fibrosis， the EZH2 inhibitors， and TCM formulas and active components targeting EZH2 has great meanings. This paper reviews the research progress in EZH2 and fibrosis， providing new ideas for the diagnosis， treatment， and drug development of fibrosis.

Expression of EZH2 and H3K27me3 predicts tumor biology of urothelial carcinoma

Background: Enhancer of zeste homolog 2 (EZH2) is one of the major epigenetic modifiers involved in the transcriptional repression of target genes through trimethylation of H3K27 (lysine 27 residue of histone H3). Deregulated expression of both EZH2 and H3K27me3 has been implicated in the biological behavior and prognostic outcome of various malignancies. Aim: To assess the role of EZH2 and H3K27me3 in the carcinogenesis of urothelial carcinoma of urinary bladder. Materials and Methods: One hundred fifty consecutive urothelial carcinoma cases of urinary bladder (54.7% high-grade) were included in this study. Immunohistochemical analysis for EZH2 and H3K27me3 was performed on whole tissue sections. A multiplication score obtained by multiplying staining intensity and proportion of positively stained neoplastic cells was used for assessment. Results: EZH2 showed a significant correlation with the tumor grade and lamina propria invasion (p < 0.001). The cases with high EZH2 expression showed a significantly high proliferative index (Mean- 32.7%; p < 0.001). In contrast, negative and low expression of H3K27me3 was significantly more common in high-grade cases (p = 0.006). The expression of H3K27me3 was significantly associated with lamina propria (p = 0.01) and deep muscle invasion (p = 0.007). EZH2 showed a significantly higher expression in the high-grade invasive areas as compared to the high-grade non-invasive areas of the same tumor (p = 0.03). Conclusions: This study establishes an important role of the key epigenetic regulators EZH2 and H3K27me3 in the pathobiology of urothelial carcinomas. Strong expression of EZH2 and weak expression of H3K27me3 are associated with higher grade, proliferative index and invasive behavior.

Effects and molecular mechanism of histone methyltransferase enhancer of zeste homolog 2 on regulating sepsis-induced T cell dysfunction / 中华危重病急救医学

Objective:To investigate the effect and mechanism of histone methyltransferase enhancer of zeste homolog 2 (EZH2) on sepsis-induced T cell dysfunction.Methods:Twenty-four male C57BL/6 mice were divided into three groups randomly: sham operated group, sepsis model group [cecum ligation and puncture (CLP)+dimethyl sulfoxide (DMSO) group] and EZH2 selective inhibitor treated group (CLP+GSK126 group), with 8 mice in each group. Sepsis murine model was reproduced by CLP. CLP+DMSO group and CLP+GSK126 group were treated with DMSO or GSK126 (10 mg/kg) respectively right after surgery through intraperitoneal injection. The mice were sacrificed 24 hours after operation, and the mesenteric lymph nodes were collected. The expression of EZH2, apoptosis rates, cell proliferation marker ki-67 antigen positive T lymphocytes (ki-67 + cell), interferon-γ positive T lymphocytes (IFN-γ + cell), programmed death receptor-1 positive T lymphocytes (PD-1 + cell) and programmed death-ligand 1 positive T lymphocytes (PD-L1 + cell) were determined by flow cytometry. Results:Compared with sham operated group, the expression of EZH2 in T lymphocytes was up-regulated on mesenteric lymph nodes of CLP+DMSO group. Compared with CLP+DMSO group, the ratio of CD3 + T lymphocytes in CLP+GSK126 group was up-regulated (0.70±0.02 vs. 0.50±0.07, P < 0.01), indicating that the EZH2 inhibitor could increase the number of T lymphocytes in lymph nodes of septic mice; the ratio of ki-67 + cells in CD4 + and CD8 + T lymphocytes in CLP+GSK126 group was increased (CD4 +: 0.74±0.05 vs. 0.63±0.04, CD8 +: 0.82±0.06 vs. 0.70±0.04, both P < 0.05), indicating that the EZH2 inhibitor could increase the ratio of T lymphocytes with high proliferative activity in lymph nodes of septic mice. However, no significant difference was found on both CD4 + and CD8 + T lymphocytes apoptosis rates in the mesenteric lymph nodes of mice between CLP+GSK126 group and CLP+DMSO group [CD4 +: (21.53±2.87)% vs. (20.48±3.21)%, CD8 +: (8.34±1.02)% vs. (7.71±1.38)%, both P > 0.05], indicating that no extra T lymphocytes apoptosis was induced by EZH2 inhibitor. Compared with CLP+DMSO group, the ratios of IFN-γ + CD4 + and IFN-γ + CD8 + T lymphocytes were increased in CLP+GSK126 group (IFN-γ +CD4 +: 0.31±0.11 vs. 0.14±0.06, IFN-γ +CD8 +: 0.30±0.10 vs. 0.13±0.06, both P < 0.05), suggesting that secretion of IFN-γ in lymph nodes by sepsis T lymphocytes was augmented after EZH2 inhibitor administration. Furthermore, compared with CLP+DMSO group, the ratio of PD-1 + cell in CD8 + T lymphocyte was down-regulated in CLP+GSK126 group (0.092±0.006 vs. 0.135±0.004, P < 0.01), suggesting that EZH2 inhibitor restrained the PD-1 expression on sepsis lymphoid node CD8 + T lymphocytes, however, it had no significant effect on PD-L1 + cells. Conclusion:EZH2, regulates sepsis-induced T lymphocyte dysfunction, possibly through modulating the expression of PD-1.

Long non-coding RNA SNHG1 promotes bladder cancer progression by upregulating EZH2 and repressing KLF2 transcription

Abstract Objective: Long Non-Coding RNAs (LncRNAs) act as an indispensable role in cancer development. The study aimed to investigate the role and mechanism of lncRNA Small Nucleolar RNA Host Gene 1 (SNHG1) in Bladder Cancer (BC) progression. Method: The expression, prognostic value, diagnostic value, and correlation of SNHG1, Enhancer of Zeste 2 polycomb repressive complex 2 subunit (EZH2), and Kruppel Like Factor 2 (KLF2) were analyzed through bioinformatics analysis. The expression was also validated in BC tissues and cell lines. Besides, their regulation and binding were tested via qPCR, Western blot, Dual-Luciferase Reporter Assay (DLRA), Argonaute RISC catalytic component 2-RNA Immunoprecipitation (AGO2-RIP), and Chromatin Immunoprecipitation (ChIP). A xenograft model in nude mice was also established. Results: SNHG1 was significantly overexpressed in BC tissues and cells. Importantly, SNHG1 was associated with poor survival, and ROC curves revealed high diagnostic values. Moreover, by CCK8, wound healing, transwell, and Western blot analysis, SNHG1 knockdown significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of BC cells. Additionally, in vivo experiments showed that silencing SNHG1 hindered tumorigenesis and tumor growth. Regarding mechanism, the results of AGO2-RIP, ChIP or DLRA showed that SNHG1 played different roles at diverse subcellular sites. In the cytoplasm, SNHG1 acted as a competing endogenous RNA for miR-137-3p to promote EZH2 expression. In the nucleus, SNHG1 could interact with EZH2 to inhibit KLF2 transcription. Conclusion: Our study elucidated that SNHG1 formed a regulatory network and played an oncogenic role in BC, which provided a novel therapeutic target for BC treatment.

Role and action mechanism of microRNA-26a targeting EZH2 in ultraviolet A-induced photoaging of human skin fibroblasts / 中华皮肤科杂志

Objective:To investigate the expression of microRNA (miR) -26a in human skin fibroblasts during photoaging induced by ultraviolet A (UVA) , and to evaluate the effect of up-or down-regulation of miR-26a expression on the methylation level of the whole genome, the target gene enhancer of zeste homolog 2 (EZH2) and cell aging.Methods:Some human skin fibroblasts were irradiated with 10 J/cm 2 UVA once a day for 7 consecutive days, RNA was extracted on days 0, 3 and 7, and real-time quantitative reverse PCR (RT-PCR) was performed to determine the expression of miR-26a; miR-26a mimics and inhibitors were transfected into fibroblasts to up-or down-regulate the expression of miR-26a respectively, and fluorescence microscopy and RT-PCR were performed to determine the expression of miR-26a and evaluate the transfection efficiency. Some human skin fibroblasts were divided into 6 groups: blank control group receiving no treatment, UVA group treated with UVA irradiation according to the above method, miR-26a mimic group transfected with miR-26a-mimics, UVA+miR-26a mimic group transfected with miR-26a-mimics followed by UVA irradiation, miR-26a inhibitor group transfected with miR-26a inhibitors, UVA+miR-26a inhibitor group transfected with miR-26a inhibitors followed by UVA irradiation. On day 7, cells in each group were collected after the end of UVA irradiation. Then, flow cytometry was performed to detect cell cycle, DNA methylation quantitative detection kit was used to detect the methylation level of whole genome, RT-PCR was conducted to determine the mRNA expression of EZH2 (a histone-lysine N-methyltransferase enzyme) , DNA methyltransferase 1 (DNMT1) and miR-26a, and Western blot analysis was performed to determine the protein expression of EZH2 and DNMT1. Statistical analysis was carried out by using one-way analysis of variance and least significant difference- t test. Results:Compared with the unirradiated control group, the expression of miR-26a gradually increased in the UVA irradiation group over time during the culture, and there was a significant difference in the expression of miR-26a between the two groups after 7 days of UVA irradiation ( t=5.295, P < 0.05) . Strong fluorescence signals were observed in the miR-26a mimic-or miR-26a inhibitor-transfected fibroblasts, suggesting a high transfection efficiency. Flow cytometry showed that the proportion of cells at G1 phase significantly differed among the blank control group, UVA group, miR-26a mimic group, UVA+miR-26a mimic group, miR-26a inhibitor group, and UVA+miR-26a inhibitor group (52.82% ± 2.56%, 78.56% ± 4.34%, 53.63% ± 3.13%, 89.52% ± 4.17%, 54.39% ± 3.86%, 65.34% ± 4.78%, respectively; F=46.728, P < 0.01) , and significantly higher in the UVA group than in the blank control group ( t=8.848, P < 0.01) , higher in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group ( t=11.922, 3.154, P < 0.01, < 0.05, respectively) , and higher in the UVA+miR-26a inhibitor group than in the miR-26a-inhibitor group ( t=3.087, P < 0.05) , but significantly lower in the UVA+miR-26a inhibitor group than in the UVA group ( t=3.547, P < 0.05) . Detection of the genome-wide methylation level showed that the methylation level ( A450 value) significantly differed among the above groups (0.676 ± 0.024, 0.323 ± 0.043, 0.506 ± 0.035, 0.169 ± 0.024, 0.602 ± 0.036, 0.422 ± 0.029, respectively, F=97.402, P < 0.01) , and significantly lower in the UVA group than in the blank control group ( P < 0.01) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.01) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.01) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . RT-PCR and Western blot analysis showed significant differences in the mRNA and protein expression of EZH2 and DNMT1 respectively among the 6 groups (both P < 0.05) , which were significantly lower in the UVA group than in the blank control group ( P < 0.05) , lower in the UVA+miR-26a mimic group than in the miR-26a mimic group and UVA group (both P < 0.05) , and lower in the UVA+miR-26a inhibitor group than in the miR-26a inhibitor group ( P < 0.05) , but significantly higher in the UVA+miR-26a inhibitor group than in the UVA group ( P < 0.05) . Conclusion:In the UVA irradiation-induced photoaging of skin fibroblasts, miR-26a expression was up-regulated, cellular proliferative activity and genome-wide methylation level decreased; up-regulation of miR-26a expression could down-regulate the expression of its target gene EZH2 and methylation-related gene DNM1, and promote cell photoaging, while down-regulation of miR-26a expression could up-regulate the expression of EZH2 and DNMT1, and inhibit cell photoaging.

A novel SIRT6 activator ameliorates neuroinflammation and ischemic brain injury

Ischemic stroke is the second leading cause of death worldwide with limited medications and neuroinflammation was recognized as a critical player in the progression of stroke, but how to control the overactive neuroinflammation is still a long-standing challenge. Here, we designed a novel SIRT6 activator MDL-811 which remarkably inhibited inflammatory response in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and primary mouse microglia, which were abolished by silencing SIRT6. RNA-seq screening identified the forkhead box C1 (

Increased EZH2 Levels in Anterior Cingulate Cortex Microglia Aggravate Neuropathic Pain by Inhibiting Autophagy Following Brachial Plexus Avulsion in Rats / 神经科学通报·英文版

After brachial plexus avulsion (BPA), microglia induce inflammation, initiating and maintaining neuropathic pain. EZH2 (enhancer of zeste homolog 2) has been implicated in inflammation and neuropathic pain, but the mechanisms by which it regulates neuropathic pain remain unclear. Here, we found that EZH2 levels were markedly upregulated during BPA-induced neuropathic pain in vivo and in vitro, stimulating pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) secretion in vivo. In rats with BPA-induced neuropathic pain, mechanical and cold hypersensitivities were induced by EZH2 upregulation and inhibited by EZH2 downregulation in the anterior cingulate cortex. Microglial autophagy was also significantly inhibited, with EZH2 inhibition activating autophagy and reducing neuroinflammation in vivo. However, this effect was impaired by inhibiting autophagy with 3-methyladenine, suggesting that the MTOR signaling pathway is a functional target of EZH2. These data suggest that EZH2 regulates neuroinflammation and neuropathic pain via a novel MTOR-mediated autophagy signaling pathway, providing a promising approach for managing neuropathic pain.

miR-143-3p regulates proliferation, migration and invasion of colon cancer RKO cells via targeting EZH2 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the molecular mechanism of miR-143-3p regulating the proliferation, migration and invasion of colon cancer RKO cells via targeting enhancer of zeste homolog 2 (EZH2). Methods: A total of 40 pairs of colon cancer tissues and corresponding para-cancerous tissues resected in the First Affiliated Hospital of Kunming Medical University from March 2015 to July 2017 were collected for this study. In addition, colon cancer cell lines (COLO320, RKO and CL-11) and normal intestinal mucosa NCM460 cells were also collected. qPCR was applied to detect the expression level of miR-143-3p in colon cancer tissues and cell lines. miR-143-3p mimics, miR-143-3p inhibitor, EZH2 siRNA and negative control plasmids were transfected into RKO cells, respectively. The effect of miR-143-3p/EZH2 axis on the proliferation, migration and invasion of RKO cells were detected by CCK-8 and Transwell assay, respectively. Western blotting was used to detect the expression level of EZH2 protein in RKO cells. The targeting relationship between miR-143-3p and EZH2 was verified by Dual luciferase reporter gene assay. Results: The expression level of miR-143-3p was downregulated in colon cancer tissues and cell lines (all P<0.01). Overexpression of miR-143-3p significantly inhibited the proliferation, migration and invasion of RKO cells (all P<0.01). Dual luciferase reporter gene assay confirmed that EZH2 was a target gene of miR-143-3p. Simultaneous knockdown of miR-143-3p and EZH2 attenuated the inhibition of EZH2 knockdown on the proliferation, migration and invasion of RKO cells. Conclusion: miR-143-3p suppresses the proliferation, migration and invasion of colon cancer cells via targetedly down-regulating EZH2.

Effect of lincRNA PVT1 associated with Enhancer of Zeste homolog 2 (EZH2) and with androgen receptor (AR) on the large-scale gene expression in LNCaP prostate cancer cells

O lincRNA PVT1 (Plasmacytoma Variant Translocation 1) é um RNA longo não codificador de proteínas (ncRNA) descrito como um oncogene sendo superexpresso em vários tipos de cânceres. LincRNA PVT1 está localizado na região genômica 8q24, também conhecida como 'gene desert'. O nível de expressão do lincRNA PVT1 está associado ao aumento do risco de câncer de próstata (PCa) e está correlacionado com os níveis de expressão do receptor de andrógeno (AR). No entanto, o mecanismo do envolvimento do lincRNA PVT1 com o AR no desenvolvimento de câncer de próstata ainda não está bem esclarecido. Aqui, nós testamos a hipótese que a formação do complexo AR-EZH2-PVT1 participa na regulação da expressão gênica em câncer de próstata, nas células LNCaP. A imunoprecipitação de ribonucleoproteínas seguida de PCR quantitativo (RIP-qPCR) revelou que o lincRNA PVT1 está associado fisicamente ao AR (12% do input) e à metiltransferase EZH2, proteína componente do complexo repressor Polycomb 2 (36% do input) sob condições suplementadas com andrógeno (+R1881). O lincRNA PVT1 também está associado fisicamente ao AR (10% de input) e à EZH2 (42% de input) em condições de privação de andrógeno (-R1881). Assim, a associação física entre lincRNA PVT1, AR e EZH2 é independente do hormônio andrógeno. Usando uma abordagem de estudo em larga-escala de perda e ganho de função, nossos resultados mostraram que o silenciamento do lincRNA PVT1 em células LNCaP na presença de andrógeno restaura a expressão parcialmente, totalmente ou causa superexpressão de 160 genes que tiveram a expressão inibida por andrógeno. Entre esses genes, destacamos genes envolvidos na regulação da diferenciação celular, em componentes da junção célula-célula, na inibição da migração e invasão celular e no desencadeamento da via apoptótica. Imunoprecipitação da cromatina seguida de PCR quantitativo (ChIP-qPCR), em cultura de células LNCaP suplementada com andrógeno sob silenciamento do lincRNA PVT1, mostrou aumento significativo na ocupação pela marca de histona ativadora H3K27Ac do promotor do gene NOV, um dos genes que tiveram sua expressão aumentada com o silenciamento de PVT1. O ChIP-qPCR também mostrou, após o silenciamento do lincRNA PVT1, um aumento significativo da marca H3K27me3 na região enhancer do gene NOV, uma característica de enhancers poised (prontos para ativação). Em conclusão, nós fornecemos a primeira evidência experimental para um mecanismo de ação do oncogene lincRNA PVT1 em células de câncer de próstata e demonstramos que sua ação inibidora da expressão afeta genes alvo que facilitam a proliferação e migração de células do câncer de próstata, sugerindo que o lincRNA PVT1 é um novo agente no complexo mecanismo de repressão transcricional envolvendo um RNA silenciador, o receptor de andrógeno (AR) e o potenciador de Zeste homólogo 2 (EZH2) no remodelamento da cromatina em células LNCaP

Long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) is an oncogene known to be overexpressed in various types of cancer. PVT1 lincRNA is located in the wellknown cancer-related genomic region 8q24, also known as 'gene desert. PVT1 lincRNA level of expression is associated with increased prostate cancer (PCa) risk and is correlated with androgen receptor (AR) expression levels. However, the mechanism of PVT1 and AR involvement in the development of prostate cancer is still unclear. Here, we tested the hypothesis that formation of the complex AR-EZH2-PVT1 participates in the regulation of gene expression in prostate cancer, in LNCaP cells. Ribonucleoprotein immunoprecipitation followed by quantitative PCR (RIP-qPCR) revealed that PVT1 lincRNA binds both the AR (12 % of PVT1 input) and the methyltransferase EZH2 from the Polycomb repressive complex 2 (36 % of input) under androgen-supplemented conditions (+R1881). PVT1 also binds both AR (10 % of input) and EZH2 (42 % of input) under androgen-deprived conditions (-R1881). Thus, PVT1 binding to AR and EZH2 is independent of the androgen hormone. Using a large-scale loss and gain of function approach, our results show that PVT1 knockdown (KD) in LNCaP in the presence of androgen restores the expression partially, fully or causes overexpression of 160 genes that are inhibited by androgen. Among these genes, we highlight genes involved in regulation of cell differentiation, in components of cell-cell junction, in inhibition of cell migration and invasion and in triggering of the apoptotic pathway. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with LNCaP cells in androgen-supplemented cultures under PVT1 lincRNA knockdown showed a significant increase in occupancy by the histone activation mark H3K27Ac of the promoter region of the NOV gene, one of the genes that had an increased expression upon PVT1 silencing. ChIPqPCR also showed a significant increase upon PVT1 lincRNA silencing of the H3K27me3 histone mark in the enhancer region of the NOV gene, a distinct feature of poised enhancers. In conclusion, we provide first experimental evidence for a mechanism of action of PVT1 lincRNA oncogene in prostate cancer cells, and show that its inhibitory action affects targetgenes that facilitate proliferation and migration of prostate cancer cells, thus suggesting PVT1 lincRNA as a novel lncRNA player in the complex mechanism of transcriptional repression involving a silencer RNA, the androgen receptor (AR) and the Enhancer of zeste homolog 2 (EZH2) in chromatin remodeling in LNCaP cells

Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

The role and relationship of EZH2, MMP-2 and MMP-9 in HCC invasion and metastasis / 西安交通大学学报(医学版)

Objective: To detect the expressions of enhancers of zeste homolog 2 (EZH2), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in hepatocellular carcinoma (HCC) tissues, analyze the correlation of EZH2, MMP-2 and MMP-9 expressions in HCC tissues with the clinicopathological factors of HCC to explore the role of EZH2 in the invasion and migration of HCC cells and the regulatory effects of EZH2 on MMP-2 and MMP-9. Methods: The expressions of EZH2, MMP-2 and MMP-9 in HCC tissues was detected by qRT-PCR. We analyzed the relationship of EZH2, MMP-2 and MMP-9 with the clinicopathological factors of HCC. Pearson correlation was used to analyze the correlation between EZH2, MMP-2 and MMP-9 expressions in HCC tissues. SMMC-7721 HCC cell lines with down-regulated EZH2 expression were constructed by small interfering RNA transfection. Transwell assay was used to observe the effects of EZH2 on invasion and migration of SMMC-7721 cells. qRT-PCR was used to detect the regulatory effects of EZH2 on MMP-2 and MMP-9 in HCC cells. Results: EZH2, MMP-2 and MMP-9 expressions were increased in HCC tissues, and they were correlated with adverse clinicopathological factors. There was a significant correlation between the expressions of EZH2 and MMP-9 in HCC tissues. Deletion of EZH2 significantly inhibited the invasion and migration of HCC cells and inhibited MMP-9 expression in HCC cells. Conclusion: EZH2, MMP-2 and MMP-9 are all closely associated with HCC progression, and they can be potential biomarkers and therapeutic targets for HCC.

lncRNA XIST regulates the malignant biological behaviors of colorectal cancer HCT-8 cells through miR-32-5p/EZH2 molecular axis / 中国肿瘤生物治疗杂志

@#Objective: To explore the mechanism of lncRNA XIST (XIST) regulating the biological behaviors of colorectal cancer HCT-8 cells via miR-32-5p/EZH2 (enhancer of Zeste homolog 2) axis. Methods:Atotal of 28 pairs of cancer tissues and corresponding para-cancerous tissues form colorectal cancer patients with complete clinical data were collected from the Colorectal and Anal Surgery, Xiangya Hospital of Central South University during July 2014 and August 2018. The expression levels of lncRNA XIST and miR-325p in colorectal cancer tissues and cell lines were detected by qPCR. The targeted relationship between lncRNA XIST, miR-32-5p and EZH2 was verified by dual luciferase reporter gene, and the expression level of EZH2 was further detected by WB. The proliferation, migration and apoptosis of HCT-8 cells were detected by CCK-8, Transwell and flow cytometry with Annexin V-FITC/PI staining, respectively. Results: lncRNAXIST was highly expressed in colorectal cancer tissues and cell lines with the highest expression in HCT-8 cells (P<0.05 or P<0.01). Dual luciferase reporter gene assay validated that lncRNA XIST negatively regulated miR-32-5p (P<0.05), and EZH2 was a target gene of miR-32-5p. Knockdown of lncRNAXIST inhibited proliferation and migration and induced apoptosis of HCT-8 cells (P<0.05 or P<0.01). Further experiments demonstrated that knockdown of lncRNA XIST up-regulated the expression of miR-32-5p and further down-regulated the expression level of EZH2, thereby inhibiting the proliferation and migration of HCT-8 cells and inducing apoptosis. Conclusion: lncRNAXIST promotes proliferation, migration and inhibits apoptosis of HCT-8 cells via miR-325p/EZH2 axis.

Effects of EZH2 inhibitor DZNeP on osteogenic differentiation of periodontal ligament stem cells / 国际生物医学工程杂志

Objective To observe the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to investigate the epigenetic regulation of EZH2 inhibitor DZNeP on osteogenic differentiation of hPDLSCs. Methods The hPDLSCs were isolated and cultured, and their proliferation under different concentrations of DZNeP (0, 1, 2, 5 and 10 μmol/L) was detected by MTT. The effects of DZNeP on osteogenic differentiation of hPDLSCs were observed by alkaline phosphatase (ALP) staining and alizarin red staining. The effect of DZNeP on the trimethylation of histone H3K27 in hPDLSCs was detected by immunofluorescence staining. Results Compared with the control group, the proliferation of hPDLSCs after 1, 2, 5 and 10 μmol/L DZNeP treatment for 48 h was significantly decreased, respectively (all P<0.05), and it was concentration-dependent. The result of ALP staining and alizarin red staining showed that DZNeP could promote the expression of early osteogenic markers ALP and the formation of advanced calcified nodules of hPDLSCs. The immunofluorescence staining result showed that the trimethylation fluorescence intensity of histone H3K27 was significantly decreased in the DZNeP group compared with the control group. Conclusions As an EZH2 inhibitor, DZNeP can inhibit the proliferation of hPDLSCs and promote the differentiation of hPDLSCs into osteoblasts in vitro, suggesting that DZNeP can be used as a potential small molecule drug for the treatment of periodontitis.

Effects of histone methyltransferase inhibitor on the polarization of peritoneal macrophages in septic mice / 中华危重病急救医学

Objective? To?investigate?the?effect?of?histone?methyltransferase?(EZH2)?inhibitor?on?the?polarization??of?peritoneal?macrophages?in?septic?mice.? Methods? Thirty-six?healthy?male?C57BL/6J?mice?were?divided?into?three?groups?by?random?number?table?method?(n?=?12):?sham?operated?group?(Sham?group),?sepsis?model?group?(CLP?group)??and?EZH2?inhibitor?treatment?group?(CLP+3-DZNeP?group).?Sepsis?animal?model?was?established?by?cecum?ligation?and?puncture?(CLP);?Sham?group?was?challenged?only?by?cecum?traction?without?ligation.?3-Deazaneplanocin?A?(3-DZNeP)?1?mg/kg?was?intraperitoneal?injected?24?hours?before?and?1?hour?after?CLP?in?CLP+3-DZNeP?group.?Eight?mice?in?each?group?were?sacrificed?at?24?hours?after?surgery.?The?levels?of?proinflammatory?cytokines?interleukin-6??(IL-6)?and?tumor?necrosis?factor-α(TNF-α)?in?peritoneal?lavatory?fluid?were?detected?by?high?throughput?liquid?protein?chip.?The?expression?levels?of?inducible?nitrogenase?(iNOS)?and?macrophage?mannose?receptor?(CD206)?were?analyzed?by?flow?cytometry.?Mouse?peritoneal?macrophages?were?isolated?and?purified?by?adherent?method,?the?protein?expressions?of?EZH2,?peroxisome?proliferator-activated?receptorγ(PPARγ)?were?detected?by?Western?Blot.?The?remaining?4?mice?? were?sacrificed?at?48?hours?after?surgery,?the?histopathological?changes?of?lung?and?kidney?tissue?were?evaluated?by?hematoxylin-eosin?(HE)?staining.? Results? Compared?with?Sham?group,?the?infiltration?of?inflammatory?cells?in?lung?and?kidney?of?the?CLP?group,?the?levels?of?IL-6?and?TNF-α?in?peritoneal?lavatory?fluid?were?significant?increased??[IL-6?(ng/L):?7?794.75±405.56?vs.?78.63±74.09,?TNF-α(ng/L):?147.25±25.19?vs.?18.20±5.03,?both?P?<?0.01],?the?percentage?of?M1?type?macrophages?was?significantly?increased?[iNOS+?F4/80+:?(13.18±8.80)%?vs.?(1.57±0.77)%,?P?<?0.05],?and?the?protein?expression?of?EZH2?was?significantly?increased?(EZH2/GAPDH:?0.84±0.11?vs.?0.11±0.03,?P?<?0.01),?while?the?protein?expression?of?PPARγ?was?significantly?decreased?(PPARγ/GAPDH:?0.09±0.01?vs.?0.27±0.09,?P?<?0.01).?Compared?with?CLP?group,?the?histopathological?changes?of?lung?and?kidney?in?CLP+3-DZNeP?group?were?significantly?alleviated,?the?levels?of?IL-6?and?TNF-α?in?peritoneal?lavatory?fluid?were?significantly?decreased?[IL-6?(ng/L):?4?207.10±876.60?vs.?7?794.75±405.56,?TNF-α(ng/L):?63.00±25.37?vs.?147.25±25.19,?both?P <?0.01?],?the?percentage?of?M1?type?macrophages?was?significantly?decreased?[iNOS+?F4/80+:?(3.64±0.89)%?vs.?(13.18±8.80)%,??P?<?0.05],?while?the?percentage?of?M2?type?macrophages?was?significantly?increased?[CD206+?F4/80+:?(17.68±5.63)%?vs.?(7.60±3.17)%,?P?<?0.01],?the?protein?expression?of?EZH2?was?significantly?decreased?(EZH2/GAPDH:?0.53±0.09?vs.?0.84±0.11,?P?<?0.05),?and?the?protein?expression?of?PPARγ?was?significantly?increased?(PPARγ/GAPDH:?0.39±0.14?vs.?0.09±0.01,?P?<?0.05).? Conclusions? Sepsis?induces?high?expression?of?EZH2?in?peritoneal?macrophages,?and?may?induce?polarization?of?M1?type?macrophages?by?inhibiting?the?expression?of?PPARγ?protein.?EZH2?inhibitor?3-DZNeP?can?lessen?the?inflammatory?cytokines?release?by?inhibiting?the?M1?type?macrophages?polarization.

mA Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2 / 基因组蛋白质组与生物信息学报·英文版

N-methyladenosine (mA), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of mA modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced mA levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. mA immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that mA was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, mA was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated mA modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.

Role of EZH2 Inhibitor Combined with Gefitinib in EGFR-TKIs Resistant Lung Cancer Cells / 中国肺癌杂志

BACKGROUND@#Lung cancer is one of the common malignant tumors that impair human health. With the development of epigenetics, the researchers found that enhancer of Zeste homolog 2 (EZH2) is highly expressed in lung cancer tissue and its expression is closely related to the prognosis. EZH2 inhibitor can also enhance the sensitivity of tumor cells to a variety of anti-tumor drugs. The purpose of this study is to investigate the effect of combination of EZH2 inhibitor and gefitinib on the proliferation, apoptosis and migration of Gefitinib-resistant lung cancer cells.@*METHODS@#PC9 and PC9/AB2 cells were used for this study. CCK-8 and EdU experiment were used to detect combined treatment on cell viability and proliferation activity; Wound healing assay and Transwell chamber experiment were used to determine the effects of combination therapy on cell migration ability; Flow cytometry was used to detect the effect of combination therapy on EZH2 and apoptosis; Western blot was used to observe the effect of combination therapy on epidermal growth factor receptor (EGFR) signaling pathway-related proteins expression.@*RESULTS@#In gefitinib-resistant cell line PC9/AB2, gefitinib combined with EZH2 inhibitor GSK343 can significantly inhibit cell viability, reduce cell migration and increase cell apoptosis. At the same time, combination therapy can significantly inhibit the expression of EZH2 and phosphorylation EGFR proteins.@*CONCLUSIONS@#The combination of EZH2 inhibitor GSK343 and gefitinib sensitize PC9/AB2 cell to gefitinib response. This study also suggests that synergistic therapy plays a role in the reversal of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) resistance in lung cancer.

Silencing of LncRNA-ANCR Promotes the Osteogenesis of Osteoblast Cells in Postmenopausal Osteoporosis via Targeting EZH2 and RUNX2

PURPOSE: This study aimed to explore the effects and mechanisms of long non-coding RNA (lncRNA) anti-differentiation non-coding RNA (ANCR) on the osteogenesis of osteoblast cells in postmenopausal osteoporosis (PMOP). MATERIALS AND METHODS: Mice models of PMOP were established. ANCR expression and intracellular calcium ions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and laser confocal microscopy, respectively. ANCR was silenced in osteoblast cells from PMOP mice by the transfection of siRNA-ANCR (si-ANCR). The proliferation and apoptosis of osteoblast cells was analyzed by MTT and flow cytometry, respectively. Alkaline phosphatase (ALP) activity and calcium nodules were examined by ALP and alizarin red staining assay, respectively. The expression of enhancer of zeste homolog 2 (EZH2), runt related transcription factor 2 (RUNX2), and OSTERIX was detected by qRT-PCR and Western blot. Furthermore, an osteogenesis model was constructed in mice, and osteoid formation was observed by hematoxylin-eosin (HE) staining. The interaction between lncRNA-ANCR and EZH2 was further identified by RNA pull-down assay. RESULTS: ANCR expression and intracellular calcium ions were increased in PMOP mice. Si-ANCR significantly increased the proliferation, ALP activity, calcium deposition of osteoblast cells and decreased apoptosis. ANCR and EZH2 were down-regulated by si-ANCR, while RUNX2 and OSTERIX were upregulated. Si-ANCR also promoted osteoid formation in mice treated with hydroxyapatite-tricalcium phosphate. In addition, ANCR specifically bound to EZH2. CONCLUSION: Silencing ANCR promotes the osteogenesis of PMOP osteoblast cells. The specific binding of ANCR with EZH2 suppressed RUNX2, thereby inhibiting osteogenesis.

Transcriptional repression by androgen receptor: Roles in castration-resistant prostate cancer / 亚洲男科学杂志(英文版)

Androgen receptor (AR), a hormonal transcription factor, plays important roles during prostate cancer progression and is a key target for therapeutic interventions. While androgen-deprivation therapies are initially successful in regressing prostate tumors, the disease ultimately comes back as castration-resistant prostate cancer (CRPC) or at the late stage as neuroendocrine prostate cancer (NEPC). CRPC remains largely dependent on hyperactive AR signaling in the milieu of low androgen, while NEPC is negative of AR expression but positive of many AR-repressed genes. Recent technological advances in genome-wide analysis of transcription factor binding sites have revealed an unprecedented set of AR target genes. In addition to its well-known function in activating gene expression, AR is increasingly known to also act as a transcriptional repressor. Here, we review the molecular mechanisms by which AR represses gene expression. We also summarize AR-repressed genes that are aberrantly upregulated in CRPC and NEPC and represent promising targets for therapeutic intervention.

Effects of EZH2 siRNA on apoptosis induction and radiosensitivity of tongue cancer Tca-8113 cells / 中华放射医学与防护杂志

Objective To investigate the effect of EZH2 on apoptosis and radiosensitivity of squamous cell carcinoma of tongue.Methods Tongue squamous carcinoma cells Tca-8113 were transfected with small interfering RNA of EZH2 (EZH2 siRNA1,EZH2 siRNA2) and its negative controls (siRNA-NC),the expression levels of EZH2 were detected by RT-PCR and Western blot and EZH2 siRNA2 was used for further studied since its better interference efficiency.The cells with siRNA transfection were irradiated with 8 Gy doses,and cell proliferation was detected by MTT,apoptosis was detected by flow cytometry,the expression of p-STAT3,STAT3 and Cleaved Caspase-3 was detected by Western blot.In addition,the cells were irradiated with 0,2,4,6,and 8 Gy to detect radiosensitivity by cell colony formation assay.Results EZH2 siRNA1 and EZH2 siRNA2 decreased the expression of EZH2 in Tca-8113 cells and EZH2 siRNA2 had a better interference efficiency (tmRNA =8.660,PmRNA ＜ 0.01;tprotein =2.883,Pprotein ＜0.05).The apoptotic rate in the EZH2 siRNA group was (29.90 ± 1.64)％,and was increased by 8 Gy irradiation to (38.17 ± 1.59) ％ (t =4.742,P ＜ 0.05).At the same time,EZH2 siRNA reduced the level of p-STAT3,but promoted the expression of Cleaved Caspase-3 protein,and enhanced the sensitivity of Tca-8113 cells to 1.668-times of control.Conclusions Interfering EZH2 could promote apoptosis,inhibit proliferation and increase radiosensitivity of squamous cell carcinoma of tongue.

Suppression on invasion and migration of human tongue squamous cell carcinoma after knocking down EZH2 expression / 中国肿瘤临床

Objective:To explore whether EZH2 can regulate the expression of miR-200b/a/429 and,thus,affect human tongue squamous cell carcinoma(TSCC).Methods:EZH2 was knocked down in TSCC lines SCC15 and UM-1 with siRNA(si-EZH2)method.The expression levels of EZH2 and epithelial mesenchymal transition related proteins were detected by Western blot.qPCR was used to determine the expression level of miR-200b/a/429 after knockdown of EZH2.Transwell and wound-healing assays were employed to detect the invasion and migration ability of tumor cells.The cytoskeleton was observed with an immunofluorescence assay.EZH2 expression in human head and neck squamous cell carcinoma(HNSCC)was detected by an immunofluorescence assay and qPCR.Results:EZH2 was significantly knocked down by siRNA, thus the expression level of miR-200b/a/429 and E-cadherin increased.While the expression of the N-cadherin,Vimentin,MMP2,and MMP9 proteins decreased;the migration and invasion of HNSCC cells in the si-EZH2 group was markedly inhibited.The EZH2 expression level in patients with lymph node metastasis in HNSCC specimens was higher than those without lymph node metastasis(P<0.01).Conclusions:EZH2 inhibits the expression of miR-200b/a/429 and promotes the invasion and migration of TSCC cells.