High EZH2 Protein Expression Is Associated with Poor Overall Survival in Patients with Luminal A Breast Cancer / 한국유방암학회지

PURPOSE: The enhancer of zeste homologue 2 (EZH2) is a catalytic subunit of the polycomb repressive complex 2, a highly conserved histone methyltransferase. EZH2 overexpression has been implicated in various malignancies, including breast cancer, where is associated with poor outcomes. This study aims to clarify nuclear EZH2 expression levels in breast cancers using immunohistochemistry (IHC) and correlate these findings with clinicopathologic variables, including prognostic significance. METHODS: IHC was performed on tissue microarrays of 432 invasive ductal carcinoma (IDC) tumors. Associations between EZH2 expression, clinicopathologic characteristics, and molecular subtype were retrospectively analyzed. The relationship between EZH2 protein expression in normal breast tissue and ductal carcinoma in situ (DCIS) was also assessed. RESULTS: High EZH2 expression was demonstrated in 215 of 432 tumors (49.8%). EZH2 was more frequently expressed in DCIS and IDC than in normal breast tissue (p=0.001). High EZH2 expression significantly correlated with high histologic grade (p<0.001), large tumor size (p=0.014), advanced pathologic stage (p=0.006), negative estrogen receptor status (p<0.001), positive human epidermal growth factor receptor 2 (HER2) status (p<0.001), high Ki-67 staining index (p<0.001), positive cytokeratin 5/6 status (p=0.003), positive epidermal growth factor receptor status (p<0.001), and positive p53 status (p<0.001). Based on molecular subtypes, high EZH2 expression was significantly associated with HER2-negative luminal B, HER2-positive luminal B, and HER2 type and triple-negative basal cancers (p<0.001). In patients with luminal A, there was a significant trend toward shorter overall survival for those with tumors having high EZH2 expression compared to those with tumors having low EZH2 expression (p=0.045). CONCLUSION: EZH2 is frequently upregulated in breast malignancies, and it may play an important role in cancer development and progression. Furthermore, EZH2 may be a prognostic marker, especially in patients with luminal A cancer.

Long Non-coding RNA HOTAIR Expression in Diffuse Large B-Cell Lymphoma: In Relation to Polycomb Repressive Complex Pathway Proteins and H3K27 Trimethylation

BACKGROUND: A long non-coding RNA hox transcript antisense intergenic RNA (HOTAIR) is involved in epigenetic regulation through chromatin remodeling by recruiting polycomb repressive complex 2 (PRC2) proteins (EZH2, SUZ12, and EED) that induce histone H3 trimethylation at lysine 27 (H3K27me3). Deregulation of c-MYC and interaction between c-MYC and EZH2 are well known in lymphomagenesis; however, little is known about the expression status of HOTAIR in diffuse large B-cell lymphomas (DLBCLs). METHODS: The expression status of PRC2 (EZH2, SUZ12, and EED), H3K27me3, c-MYC, and BCL2 was analyzed using immunohistochemistry (n = 231), and HOTAIR was investigated by a quantification real-time polymerase chain reaction method (n = 164) in DLBCLs. RESULTS: The present study confirmed the positive correlation among PRC2 proteins, H3K27me3, and c-MYC in DLBCLs. Expression level of HOTAIR was also positively correlated to EZH2 (p < .05, respectively). Between c-MYC and HOTAIR, and between c- MYC/BCL2 co-expression and HOTAIR, however, negative correlation was observed in DLBCLs (p < .05, respectively). High level of H3K27me3 was determined as an independent prognostic marker in poor overall survival (hazard ratio, 2.0; p = .023) of DLBCL patients. High expression of HOTAIR, however, was associated with favorable overall survival (p = .004) in the univariate analysis, but the impact was not significant in the multivariate analysis. The favorable outcome of DLBCL with HOTAIR high expression levels may be related to the negative correlation with c- MYC expression or c-MYC/BCL2 co-expression. CONCLUSIONS: HOTAIR expression could be one of possible mechanisms for inducing H3K27me3 via EZH2-related PRC2 activation, and induced H3K27me3 may be strongly related to aggressive DLBCLs which show poor patient outcome.

Expression of enhancer of zeste homolog 2 in esophageal squamous cell carcinoma and its prognostic value in postoperative patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of enhancer of zeste homolog 2 (EZH2) in esophageal squamous cell carcinoma and its association with the prognosis of postoperative patients.</p><p><b>METHODS</b>Surgical specimens were obtained from 102 patients with esophageal squamous cell carcinoma undergoing radical resection in our hospital from 1996 to 2006. Immunochemistry was employed to examine EZH2 protein expressions in the specimens, including 102 carcinoma tissue specimens, 30 adjacent tissue specimens and 30 normal esophageal tissue specimens. The expression levels of EZH2 were analyzed in relation to the clinicopathological parameters of the patients including gender, age, tumor differentiation, TNM, and lymph node metastasis. The postoperative patients were followed up to analyze the association of EZH2 expression with the clinical outcomes.</p><p><b>RESULTS</b>The esophageal squamous cell carcinoma tissue showed a higher EZH2 expression than the adjacent and normal esophageal tissues. EZH2 expression was higher in poorly differentiated carcinoma than in well differentiated tissue, and also higher in cases with lymph node metastasis than those without; the expression was higher in TNM stage II/III patients than in stage I patients but lower than in stage IV patients. The patients with low EZH2 expression was found to have a longer survival time than those with high EZH2 expression (P<0.05).</p><p><b>CONCLUSION</b>EZH2 plays an important role in the differentiation and metastasis of esophageal squamous cell carcinoma, and a high EZH2 expression is associated with a poor outcome in the the postoperative patients.</p>

miRNA-101 inhibits the expression of the enhancer of zeste homolog 2 in androgen-independent prostate cancer LNCaP cell line / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effect of miRNA-101 on the expression of the enhancer of zeste homolog 2 (EXH2) in human androgen-independent prostated cancer LNCaP cells.</p><p><b>METHODS</b>We divided LNCaP cells into a blank control, a negative control, and a miRNA-l01 transfection group, constructed the vector by transfecting synthetic miRNA-101 mimics into the LNCaP cells, and evaluated the efficiency of transfection by fluorescence microscopy. Then we determined the expression level of EZH2 mRNA by qRT-PCR in the three groups of cells and that of the EZH2 protein in the negative control and transfection groups by Western blot.</p><p><b>RESULTS</b>Green fluorescence signals were observed in over 70% of the LNCaP cells in the transfection group after 24 hours of transfection. At 72 hours, the expression of miRNA-101 was significantly upregulated in the transfected cells (P < 0.01), that of EZH2 mRNA was remarkably lower in the transfection group (0.01 ± 0.10) than in the blank control (0.95 ± 0.40) and negative control (0.86 ± 0.30) groups (both P < 0.01), and that of the EZH2 protein was increased in the negative control but decreased in the transfection group with the extension of culture time.</p><p><b>CONCLUSION</b>miRNA-101, with its inhibitory effect on the expression of EZH2 in LNCaP cells, is a potential biotherapeutic for prostate cancer.</p>

Driver mutations of cancer epigenomes

Epigenetic alterations are associated with all aspects of cancer, from tumor initiation to cancer progression and metastasis. It is now well understood that both losses and gains of DNA methylation as well as altered chromatin organization contribute significantly to cancer-associated phenotypes. More recently, new sequencing technologies have allowed the identification of driver mutations in epigenetic regulators, providing a mechanistic link between the cancer epigenome and genetic alterations. Oncogenic activating mutations are now known to occur in a number of epigenetic modifiers (i.e. IDH1/2, EZH2, DNMT3A), pinpointing epigenetic pathways that are involved in tumorigenesis. Similarly, investigations into the role of inactivating mutations in chromatin modifiers (i.e. KDM6A, CREBBP/EP300, SMARCB1) implicate many of these genes as tumor suppressors. Intriguingly, a number of neoplasms are defined by a plethora of mutations in epigenetic regulators, including renal, bladder, and adenoid cystic carcinomas. Particularly striking is the discovery of frequent histone H3.3 mutations in pediatric glioma, a particularly aggressive neoplasm that has long remained poorly understood. Cancer epigenetics is a relatively new, promising frontier with much potential for improving cancer outcomes. Already, therapies such as 5-azacytidine and decitabine have proven that targeting epigenetic alterations in cancer can lead to tangible benefits. Understanding how genetic alterations give rise to the cancer epigenome will offer new possibilities for developing better prognostic and therapeutic strategies.

Regulation and Role of EZH2 in Cancer / Journal of the Korean Cancer Association, 대한암학회지

Polycomb repressive complex 2 (PRC2) is the epigenetic regulator that induces histone H3 lysine 27 methylation (H3K27me3) and silences specific gene transcription. Enhancer of zeste homolog 2 (EZH2) is an enzymatic subunit of PRC2, and evidence shows that EZH2 plays an essential role in cancer initiation, development, progression, metastasis, and drug resistance. EZH2 expression is indeed regulated by various oncogenic transcription factors, tumor suppressor miRNAs, and cancer-associated non-coding RNA. EZH2 activity is also controlled by post-translational modifications, which are deregulated in cancer. The canonical role of EZH2 is gene silencing through H3K27me3, but accumulating evidence shows that EZH2 methlyates substrates other than histone and has methylase-independent functions. These non-canonical functions of EZH2 are shown to play a role in cancer progression. In this review, we summarize current information on the regulation and roles of EZH2 in cancer. We also discuss various therapeutic approaches to targeting EZH2.

Clinical significance of expression of enhancer of zeste homolog 2 and 53 in cervical squamous cell carcinoma and intraepithelial neoplasia / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression and clinical significance of enhancer of zeste homolog 2 (EZH2) and p53 proteins in cervical squamous cell carcinoma (SCC) and cervical intraepithelial neoplasia (CIN).</p><p><b>METHODS</b>The expression and distribution of EZH2 and p53 were determined with reference to clinicopathological features and patient survival. 168 cervical SCC, 19 CINII, 35 CINIII patients and 30 normal control cases were collected for immunohistochemical analysis.</p><p><b>RESULTS</b>The expression of EZH2 in the normal cervix, CIN and SCC was 6.7% (2/30), 37.0% (20/54) and 75.6% (127/168), respectively, with a significant difference between them (P < 0.05). The expression of p53 was 3.3% (1/30), 20.4% (11/54) and 39.3% (66/168), respectively, also (P < 0.05). In the 168 SCC cases, the positive rate of EZH2 in the cases with lymph node metastasis was 82.9%, and that of p53 was 45.7%; the positive rate of EZH2/p53 protein expression in the cases with negative lymph nodes was 70.4%, and that of p53 was 34.7%, with a significant difference between the two subgroups (P < 0.05). Among the 143 followed-up SCC patients, the EZH2(+) p53(+) cases had a progression-free survival of (51.3 ± 3.8) months, that of EZH2(+) or p53(+) cases was (66.1 ± 2.0) months, and that of EZH2(-) p53(-) cases was (71.3 ± 1.9) months, with a very significant difference among the three subgroups (P < 0.001). The overall survival times of EZH2(-) p53(-), EZH2(+) or p53(+), and EZH(2+) p53(+) cases were (72.9 ± 1.1), (68.6 ± 1.8), and (57.4 ± 3.4) months, respectively, with a significant difference among the three subgroups (P < 0.001). The multivariate analysis showed that EZH2 expression, lymph node metastasis and tumor staging were independent prognostic factors.</p><p><b>CONCLUSIONS</b>Both EZH2 and p53 proteins may play important roles in the occurrence and development of cervical squamous cell carcinoma. There is a close relationship between the expression of both EZH2 and p53 proteins and the prognosis of SCC patients.</p>

Screening of novel miRNAs targeting EZH2 3' untranslated region using lentivirus miRNAs library and their expressions in breast cancer cells and tissues / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen novel miRNAs targeting EZH2 3' untranslated region (UTR) in recombinational MCF-7 breast cancer cells over-expressing EZH2 3' UTR and quantitative analyze the expressions of the screened miRNA in breast cancer cells and tissues.</p><p><b>METHODS</b>A lentiviral library was transfected into the recombinant cell line MCF-7. The cells were screened with cytotoxic agents before extraction of the genome for amplification of the miRNA precursors using PCR. The screened miRNAs were identified with sequence analysis and their expressions were analyzed quantitatively with real-time PCR in breast cancer cells and tissues.</p><p><b>RESULTS</b>Seven miRNAs were screened from the recombinant MCF-7 cells, namely miR-15b, miR-16-2, miR-181b2, miR-217, miR-224, miR-329-1, and miR-487b, all of which failed to be predicted by bioinformatics software. Real-time PCR showed that miR-217, miR-329-1, and miR-487b were over-expressed in MCF-7 cells, and the expression of miR-15b and miR-16-2 was significantly increased in cancer tissues compared with the adjacent tissues (P<0.05).</p><p><b>CONCLUSION</b>Novel targeted miRNAs that can not be predicted by bioinformatics software were successfully screened from MCF-7 breast cancer cells over-expressing EZH2 3' UTR. These miRNAs are expressed differentially between normal breast cells and breast cancer tissues.</p>

Expression of a constitutively active prolactin receptor causes histone trimethylation of the p53 gene in breast cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Prolactin (PRL) is a pituitary polypeptide hormone characterized by multiple biological actions including stimulation of growth in the prostate and formation of secretory alveoli and stimulation of milk protein gene expression in the mammary gland. PRL exerts its effect by dimerizing its receptor (PRLR) on the plasma membrane and regulating gene expression through the JAK-Stat signal pathway. We have previously described a natural variant of the PRLR in which the S2 subdomain of the extracellular domain is missing (Delta S2). Delta S2 PRLRs are dimerized in the absence of PRL and have constitutive activity in the promotion of breast cancer cell growth. Enhancer of zeste homolog 2 (EZH2), as one of the histone-modifying enzymes, is a key factor regulating gene expression by epigenetic modification. We hypothesized that these constitutive activated Delta S2 PRLRs played a pathogenic role in breast cancer in part through alterations in the expression of EZH2 and the trimethylation of histone 3 on lysine 27 (H3K27Me3).</p><p><b>METHODS</b>In order to verify the clinical significance and to establish the link between Delta S2 PRLR expression and epigenetic change, EZH2, H3K27Me3, and Delta S2 PRLR were detected in both normal and cancerous human breast tissues. Also, overexpression of Delta S2 PRLR in breast epithelial cells was achieved by infection with adenovirus carrying the cDNA. Western blotting and chromatin immunoprecipitation (ChIP assay) and acid histone extraction were applied to detect the expression of EZH2 and the trimethylation of histone 3, respectively.</p><p><b>RESULTS</b>In breast tissue, higher EZH2 expression and higher H3K27Me3 were found associated with higher Delta S2 expression in breast cancer samples. In breast epithelial cells, overexpression of Delta S2 PRLR increased EZH2 methyltransferase mRNA and protein, induced EZH2 methyltransferase recruitment to chromatin, increased the trimethylation of H3K27Me3, and decreased the expression of p53 gene.</p><p><b>CONCLUSIONS</b>Delta S2 PRLR plays an important pathogenic role in breast cancer through epigenetic modification. Elevated expression of Delta S2 PRLR, achieved by alternate splicing of the pre-mRNA of the full-length form, is a new mechanism contributing to human breast cancer.</p>

Bivalent histone modifications during tooth development / 国际口腔科学杂志·英文版

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

EZH2, an epigenetic driver of prostate cancer

The histone methyltransferase EZH2 has been in the limelight of the field of cancer epigenetics for a decade now since it was first discovered to exhibit an elevated expression in metastatic prostate cancer. It persists to attract much scientific attention due to its important role in the process of cancer development and its potential of being an effective therapeutic target. Thus here we review the dysregulation of EZH2 in prostate cancer, its function, upstream regulators, downstream effectors, and current status of EZH2-targeting approaches. This review therefore provides a comprehensive overview of EZH2 in the context of prostate cancer.

Role of co-expression of c-Myc, EZH2 and p27 in prognosis of prostate cancer patients after surgery / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>c-Myc, EZH2 and p27 were defined to modulate the behavior of prostate cancer with pro-tumoral or anti-tumoral effects and had ability in predicting prostate cancer progression, but the research of their co-expression value of prognosis is rarely. This study aimed to investigate the prognostic value of combining tri-marker together in patients with intermediate-risk prostate cancer after surgery.</p><p><b>METHODS</b>Expression levels of c-Myc, EZH2 and p27 in 129 patients with intermediate-risk prostate cancer were assessed using immunohistochemistry in a semi-quantitative manner. The expression profiles of these three markers were analyzed and investigated for association with biochemical recurrence.</p><p><b>RESULTS</b>In all, fifty of 129 cases experienced biochemical recurrence during a median follow-up time of 31 months (range, 6 - 60 months). Of these relapse patients, one case without and 10 cases with any single positive marker were observed; 39 cases were detected with any two or all three positive markers (22 cases with any two and 17 cases with all three positive markers). Survival analysis showed that patients with over-expression of c-Myc or EZH2, and lower expression of p27 manifested significantly higher biochemical recurrence rates. Subsequent multivariate analysis revealed that c-Myc, EZH2 and p27 expression statuses showed potential in predicting relapse, respectively. Notably, combining three markers together as a "composite index" (0 or 1, vs. 2 or 3 positive markers) provided powerful prognostic value (HR 6.57, 95% CI 3.02 - 14.31, P < 0.001). There was a significant difference between the patient subgroups with 0 or 1 and those with 2 or 3 positive markers expression statuses, and tri-marker composite index was an independent risk factor for predicting relapse in patients with intermediate-risk prostate cancer after surgery.</p><p><b>CONCLUSION</b>Composite index of c-Myc, EZH2, and p27 can be valued as powerful prognosis parameter for intermediate-risk prostate cancer patients after the surgery, and postoperative adjuvant therapy can be adopted accordingly.</p>

Expression of enhancer of zeste homolog 2 in esophageal squamous cell carcinoma and its prognostic value in postoperative patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of enhancer of zeste homolog 2 (EZH2) in esophageal squamous cell carcinoma and its association with the prognosis of postoperative patients.</p><p><b>METHODS</b>Surgical specimens were obtained from 102 patients with esophageal squamous cell carcinoma undergoing radical resection in our hospital from 1996 to 2006. Immunochemistry was employed to examine EZH2 protein expressions in the specimens, including 102 carcinoma tissue specimens, 30 adjacent tissue specimens and 30 normal esophageal tissue specimens. The expression levels of EZH2 were analyzed in relation to the clinicopathological parameters of the patients including gender, age, tumor differentiation, TNM, and lymph node metastasis. The postoperative patients were followed up to analyze the association of EZH2 expression with the clinical outcomes.</p><p><b>RESULTS</b>The esophageal squamous cell carcinoma tissue showed a higher EZH2 expression than the adjacent and normal esophageal tissues. EZH2 expression was higher in poorly differentiated carcinoma than in well differentiated tissue, and also higher in cases with lymph node metastasis than those without; the expression was higher in TNM stage II/III patients than in stage I patients but lower than in stage IV patients. The patients with low EZH2 expression was found to have a longer survival time than those with high EZH2 expression (P<0.05).</p><p><b>CONCLUSION</b>EZH2 plays an important role in the differentiation and metastasis of esophageal squamous cell carcinoma, and a high EZH2 expression is associated with a poor outcome in the the postoperative patients.</p>

EZH2 gene silenced by siRNA suppresses the growth and invasion of endometrial carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects on cell proliferation and invasion as well as molecular basis after suppressing EZH2 expression in endometrial carcinoma cells by using siRNAs.</p><p><b>METHODS</b>RT-PCR was used to examine the expression of EZH2 in endometrial carcinoma and their paracancerous tissues. SiRNAs targeting to EZH2 were transfected to endometrial carcinoma cells, and MTT, FACS, and boyden assays were utilized to examine cell proliferation, cell cycle change, and cell invasion. Finally, the molecular mechanisms of EZH2 on cell function alteration were investigated.</p><p><b>RESULTS</b>Compared with paracancerous tissues, increased expression trend of EZH2 mRNA was showed in endometrial carcinoma tissues. Further, knocking down EZH2 expression inhibited cell growth, cell cycle transition from G1 to S phase, and cell invasion ability. Molecular basis indicated that suppression of EZH2 downregulated the expression of E2F1 and MMP9 and upregulated tumor suppressor p21 expression.</p><p><b>CONCLUSION</b>EZH2 expression is increased in endometrial carcinoma tissues. Knocking down EZH2 expression suppresses the cell growth, cell cycle transition and cell invasion by downregulated E2F1 and MMP9, and upregulated tumor suppressor p21 expression.</p>

Clinical significance of common leukemia gene mutations in patients with acute promyelocytic leukemia / 中国实验血液学杂志

This study was aimed to explore whether multiple common gene mutations of leukemia synergistically involved in acute promyelocytic leukemia (APL) pathogenesis, and to investigate their relevance to clinical features, cytogenetics and molecular risk stratification. 84 specimens of admitted de novo APL patients from February 2005 to October 2010 were collected, the gene mutations of bone marrow mononuclear cells and clinical features of mutation-positive patients were analyzed by genomic DNA-PCR. The results indicated that the prevalence of mutations was 60.7% (51/84), in which the mutations with the highest incidence were found as FLT3-ITD, reaching 27.4% (23/84). Next, there were 12 cases WT1 mutation, 9 for FLT3-TKD, 7 for TET2, 5 for N-RAS, 4 for ASXL1, 2 for EZH2 mutation and 1 positive case in MLL-PTD, IDH1 and CBL mutation respectively. No mutation was found in other JAK1, DNMT3, c-Kit, NPM1, IDH2, RUNX1 and JAK2 (V617F) common leukemia-related genes. Combined analysis with clinical data demonstrated that the patients with FLT3-ITD mutation displayed higher white blood cell counts, while the patients with N-RAS mutation showed lower platelet counts. Overall survival of these patients was obviously shorten as compared with patients with wild-type. This difference between mutant and wild-type of all above mentioned cases was statistically significant (P < 0.05). The difference between APL with simple t (15;17) and additional abnormal karyotype was not statistically significant. It is concluded that the FLT3-ITD mutation is recurrent genetic change in APL, and together with N-RAS mutation indicates poor prognosis. Additional abnormal karyotype does not associate with prognosis of APL.

Expression of EZH2 in breast cancer and its clinicopathological significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of EZH2 in breast cancer and its clinicopathological significance.</p><p><b>METHODS</b>Eighty cases of invasive breast cancer with complete clinlcpathological data and follow-up information were enrolled in the study. The expressions of ER, PR, HER-2, CK5/6, CK14, EGFR and EZH2 proteins were detected by immunohistochemistry (IHC). The relationship of EZH2 expression with molecular subtypes and prognosis of breast cancer was analyzed.</p><p><b>RESULTS</b>In 80 cases of breast cancer, there were 49 cases of Luminal subtype, 8 cases of HER-2(+) subtype, 20 cases of Basal-like subtype and 3 cases of Normal breast-like subtype. The molecular subtypes of breast cancer were associated with menopausal status of patients. Luminal subtype had the highest overall survival rate, while the Basal-like carcinomas were associated with the lowest survival (P< 0.05). The overall EZH2-positive expression rate was 45.00% and the expression was correlated with axillary lymph node metastasis, histological grading and clinical staging of breast cancer (P < 0.05). The EZH2-positive expression rates in Luminal subtype, HER-2(+) subtype and Basal-like subtype of breast cancers were 34.6%,50.0% and 70.0%, respectively (P <0.05).</p><p><b>CONCLUSION</b>Positive expression of EZH2 may indicate a poor prognosis of breast cancer patients and it might be used as a novel therapeutic target for breast cancer of Basal-like subtype.</p>

Polycomb group proteins and their roles in regulating stem cell development / 中国医学科学院学报

Polycomb group (PcG) proteins are a family of epigenetic regulators responsible for the repression of genes in proliferation and differentiation of stem cells. PcG protein complex consists of two important epigenetic regulators: PRC1 (polycomb repressive complex 1) and PRC2 (polycomb repressive complex 2). In order to further understand the functions of PcG proteins in stem cell growth and differentiation, we review the PcG protein composition, PcG protein localization in the target gene, PcG protein recruitment, and the functions of PcG proteins in the development of stem cells.

Effects of enhancer of zeste homolog (EZH2) downregulation on the proliferation and invasion of nasopharyngeal carcinoma cell and the possible mechanisms / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of the enhancer of zeste homolog 2 (EZH2) gene on cell growth and invasion of the nasopharyngeal carcinoma (NPC).</p><p><b>METHODS</b>Recombinant lentivirus vector for shRNA delivery of EZH2 was constructed and transfected into 293FT cells. After collecting the viral particles, the NPC cell line 5-8F cells were transfected. The effects of EZH2 silence on cell proliferation and cell cycle were detected using MTT assay, plate colony formation assay and flow cytometry. The migration and invasion of 5-8F cells were determined by wound healing assay and matrigel invasion assay, respectively. The expressions of EZH2 and epithelial-mesenchymal transition (EMT)-related markers at mRNA and protein levels were examined by real-time PCR and Western blot respectively.</p><p><b>RESULTS</b>The expressions of EZH2 mRNA and protein in the transfected 5-8F cells were obviously reduced. MTT assay showed that EZH2 downregulation significantly inhibited the growth of 5-8F/shEZH2 cells (P < 0.001). Colony formation rate (84.44%) of 5-8F/shEZH2 cells was lower than control (31.56%, P = 0.001). Cell cycle analysis showed that most 5-8F/shEZH2 cells were arrested in G0/G1 phase, with a very low ratio of cells in S phase. Wound healing assay indicated that the migration ability of cells silencing EZH2 decreased significantly, and the 48-hour relative migration distance of 5-8F/ShEZH2 cells and control cells was 0.58 ± 0.05, and 0.81 ± 0.02, respectively (P < 0.000). Matrigel invasion assay, showed the invasive capacity of cells silencing EZH2 was significantly inhibited, with less penetrating cells (72.23 ± 4.08) compared to control (150.95 ± 16.27), P < 0.000. The mRNA expressions of epithelial markers E-cadherin and Keratin 18 in the cells silencing EZH2 increased by 177% and 158% respectively, and the mRNA expressions of mesenchymal markers β-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot analysis.</p><p><b>CONCLUSION</b>EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro, which might be mediated by inducing EMT.</p>

Construction of a recombinant shRNA expression vector targeting EZH2 gene and its inhibitory effect on colon adenocarcinoma SW480 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct a recombinant short hairpin RNA (shRNA) expression vector targeting EZH2 gene, and to determine its effect on the proliferation of colon adenocarcinoma SW480 cells.</p><p><b>METHODS</b>The DNA sequence with short hairpin structure was designed according to the EZH2 cDNA sequence and cloned into PGFP-V-RS vector to construct a recombinant expression vector silencing EZH2 gene. After identification, the shRNA-expressing vector was then transfected into SW480 cells. RT-PCR and Western blot were used to detect the inhibitory effect at both mRNA and protein levels. MTT was used to detect cell viability due to the alteration of EZH2 gene activity.</p><p><b>RESULTS</b>At 48 h after transfection, the expression of EZH2 mRNA in the gene silencing group and negative control group were 0.339 ± 0.013 and 1.968 ± 0.072, respectively. The expression of EZH2 protein in the gene silencing group and negative control group were 0.229 ± 0.008 and 1.168 ± 0.053, respectively. The expression of EZH2 in the gene silencing group was significantly lower than that in the negative control group (P < 0.01, P < 0.05). At 48 and 72 h after transfection, the inhibition rate of cell growth in the gene silencing group was 30.7% and 25.9%, respectively, indicating that the cell growth was significantly inhibited in comparison with that in the blank control group (P < 0.05).</p><p><b>CONCLUSIONS</b>A recombinant shRNA expression vector targeting EZH2 gene has been successfully constructed in this study, with a significant inhibitory effect on the proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.</p>

Overexpression of enhancer of zests homolog 2 in lymphoma / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>This article aimed to review the biological characteristics of enhancer of zests homolog 2 (EZH2), and the transcriptional repression mechanism of action of EZH2 in tumors, particularly in the progression of lymphoma.</p><p><b>DATA SOURCES</b>The data cited in this review were mainly obtained from the articles listed in PubMed and HighWare that were published from March 2004 to April 2012. The search terms were "enhancer of zests homolog 2", "polycomb group", and "lymphoma".</p><p><b>STUDY SELECTION</b>Articles regarding the mechanism of EZH2 in post-transcriptional modification, functions of polycomb group proteins, and the roles of EZH2 in lymphoma were selected.</p><p><b>RESULTS</b>EZH2 acts as oncogene and involved in many kinds of tumors. Moreover, it plays an important role in tumorigenesis and lymphomagenesis by promoting the proliferation and aggressiveness of neoplastic cells, facilitating malignant tumor cell diffusion, and mediating transcriptional silencing.</p><p><b>CONCLUSION</b>EZH2 mediated transcriptional repression through its methyltransferase activity at the chromatin level has certain influence on lymphoma, and there might exist a therapeutic window for the development of new agents and identification of novel diagnostic markers based on EZH2.</p>