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.

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

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.

Prognostic Value of EZH2,Ki-67 and CD34 Expression in Patients with Surgically Treated Colorectal Cancer / 中山大学学报(医学科学版)

[Objective]To evaluate the expression of enhancer of zeste homolog 2(EZH2),Ki-67 and intratumoral microvaseu?lar density(iMVD)in human colorectal cancer(CRC),and discuss their relationship with the biological behavior and prognosis.[Methods]The expression of EZH2,Ki-67 and iMVD(labeled by CD34 protein)was measured with immunohistochemical MaxVision method in CRC patients followed up over 5 years.[Results]Positive expression rates of EZH2,Ki-67 and iMVD were 40.91%(45/110),57.27%(63/110)and 46.36%(51/110),respectively. EZH2 expression was positively correlated with distant metastasis(P=0.024). Ki-67 expression was positively correlated with tumor size,infiltration depth,AJCC stage and differentiation(P=0.033~0.015). The iMVD expression was positively correlated with infiltration depth ,AJCC stage and differentiation (P=0.016~0.034). Spearman correlation analysis showed that EZH2 expression was positively correlated with Ki-67 expression(r=0.195,P=0.041), whereas negatively correlated with iMVD expression. Ki-67 expression had a positive correlation with iMVD(r=0.213,P=0.025). Multivariate analysis suggested that the EZH2 expression was an adverse independent factor for survivals of CRC patients(HR 1.965, 95%CI:1.019-3.789,P=0.044 for OS).[Conclusion]EZH2 expression was significantly correlate with the proliferation of tumor cells and may plays an important role in the metastasis and prognosis of CRC.