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1.
Article in Chinese | WPRIM | ID: wpr-921550

ABSTRACT

Objective To investigate the expression and correlation of Runt-related transcription factor 3(RUNX3)and enhancer of zeste homolog 2(EZH2)in rectal cancer,and to reveal the relationship between the expression of RUNX3 and EZH2 and the sensitivity of XELOX regimen to neoadjuvant chemotherapy in locally advanced rectal cancer patients. Methods The carcinoma and paracancerous tissues of 31 patients with rectal adenocarcinoma and no preoperative antitumor therapy were selected as cancer group and paracancer group,respectively.The relative mRNA levels of RUNX3 and EZH2 in the two groups were measured by real-time quantitative reverse transcription-polymerase chain reaction,and the protein levels were determined by immunohistochemical assay.The expression of RUNX3 and EZH2 was compared between cancer tissue and paracancerous tissue.The pre-treatment wax blocks of 26 patients with locally advanced rectal cancer who received 3 cycles of XELOX regimen as neoadjuvant chemotherapy before surgery were selected as the pre-neoadjuvant therapy group,and the postoperative pathological wax blocks were selected as the post-neoadjuvant treatment group.Tumor regression grade(TRG)was determined to evaluate the efficacy of neoadjuvant therapy.Immunohistochemical assay was used to detect the protein levels of RUNX3 and EZH2 in the two groups,and then the relationship between the expression patterns of the two proteins and the efficacy of neoadjuvant chemotherapy was analyzed. Results Compared with paracancerous tissue,the cancer tissue showed down-regulated mRNA level and reduced positive protein expression rate of RUNX3,while up-regulated mRNA level(


Subject(s)
Core Binding Factor Alpha 3 Subunit/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Humans , Neoadjuvant Therapy , Rectal Neoplasms/drug therapy , Transcription Factor 3
2.
Article in Chinese | WPRIM | ID: wpr-880139

ABSTRACT

OBJECTIVE@#To investigate the predictive value of methyltransferase EZH2 expression level on the clinical efficacy and long-term prognosis of patients with primary gastrointestinal diffuse large B-cell lymphoma (PGI-DLBCL).@*METHODS@#161 patients with newly treated PGI-DLBCL in our hospital from August 2013 to July 2019 were selected. The expression level of EZH2 protein was detected by immunohistochemistry, and the short-term efficacy and long-term survival differences of patients with different levels of EZH2 were compared. The predictive values of EZH2 expression level on the short-term efficacy and long-term prognosis of PGI-DLBCL patients were analyzed by Log-rank test and COX risk proportional regression model. Chi-square test and Logistic regression analysis were used to analyze the influencing factors of EZH2 expression level.@*RESULTS@#The complete response (CR) and overal response(OR) rates of those with high EZH2 expression were significantly lower than those with low EZH2 expression (P<0.001). The median OS and PFS of EZH2 high-level and low-level expression group was 37, 31 months and 49, 42 months, respectively. The cumulative OS and PFS rates of the high-level expression group were significantly lower than those of the low-level expression group, and the differences were statistically significant (P<0.05). The high expression levels of H3K27me3, EZH2, BCL-2, BCL-6, c-MYC were closely related to the shortening of OS and PFS, while the high expression level of Ki-67 was closely related to the shortening of OS (P<0.05), of which the high expression levels of H3K27me3, EZH2, BCL-2, and BCL-6 were independent risk factors for shortening of OS and PFS. The expression level of EZH2 was positively correlated with the expression level of H3K27me3, BCL-6, c-MYC and Ki-67 (r=0.741, r=0.837, r=0.809, r=0.772), and the high expression levels of H3K27me3, BCL-6 and Ki-67 were independent factors influencing the high expression of EZH2.@*CONCLUSION@#In patients with PGI-DLBCL, the high expression of EZH2 significantly reduces the short-term CR and OR rates, which is an independent risk factor for the shortening of long-term OS and PFS rates, and it is independently related to the high expression of H3K27me3 and BCL6.


Subject(s)
Enhancer of Zeste Homolog 2 Protein , Humans , Immunohistochemistry , Lymphoma, Large B-Cell, Diffuse , Prognosis , Remission Induction , Retrospective Studies , Treatment Outcome
3.
Biol. Res ; 53: 18, 2020. tab, graf
Article in English | LILACS | ID: biblio-1124204

ABSTRACT

BACKGROUND: Cisplatin resistance (DDP-resistance) remains one of the major causes of poor prognosis in females with ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of cellular processes, including chemoresistance. The aim of this study was to explore the role of HOX transcript antisense RNA (HOTAIR) in DDP-resistant ovarian cancer cells. METHODS: DDP-resistant ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. Real-time PCR, western blot, dual-luciferase reporter assay, and flow cytometry were then used to evaluate the effect of HOTAIR/miR-138-5p axis on chemoresistance of DDP-resistant ovarian cancer cells to DDP. RESULTS: We found that HOTAIR was upregulated in DDP-resistant cells, while miR-138-5p was downregulated. Knockdown of HOTAIR increased the expression of miR-138-5p in DDP-resistant cells and miR-138-5p is directly bound to HOTAIR. Upregulation of miR-138-5p induced by HOTAIR siRNA or by its mimics enhanced the chemosensitivity of DDP-resistant cells and decreased the expression of EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) and SIRT1 (sirtuin 1). Furthermore, the HOTAIR silencing-induced chemosensitivity of DDP-resistant cells was weakened by miR-138-5p inhibitor. CONCLUSIONS: These data demonstrate that HOTAIR acts as a sponge of miR-138-5p to prevent its binding to EZH2 and SIRT1, thereby promoting DDP-resistance of ovarian cancer cells. Our work will shed light on the development of therapeutic strategies for ovarian cancer treatment.


Subject(s)
Humans , Female , Ovarian Neoplasms/genetics , Cisplatin/pharmacology , Drug Resistance, Neoplasm/genetics , RNA, Long Noncoding/genetics , Gene Expression Regulation, Neoplastic/drug effects , Up-Regulation , Apoptosis/drug effects , MicroRNAs/antagonists & inhibitors , Cell Line, Tumor , Gene Knockout Techniques/methods , Sirtuin 1/antagonists & inhibitors , Real-Time Polymerase Chain Reaction , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors
4.
Article in Chinese | WPRIM | ID: wpr-827196

ABSTRACT

OBJECTIVE@#To explore the expression and clinical significance of EZH2 in DLBCL patients accompanied by HBV infection.@*METHODS@#The clinicopathological data of 59 patients with DLBCL accompanied by HBV infection in our hospital from February 2015 to October 2017 were analyzed retrospectively. The patients were divided into HBV negative and HBV positive groups by serological testing before surgery. The expression of EZH2 was detected by immumohistochemical staining, and the clinicopathological characteristics and survival were analyzed and compared between these two groups.@*RESULTS@#There were 30 patients (50.8%) in the HBV negative group and 29 patients (49.2%)in the HBV positive group. The differences of age, LDH level and IPI score between two groups were statistically significant (P<0.05). The expression of EZH2 in HBV- positive group was significantly higher than that in the HBV- negative group (P<0.05), where the expression of EZH2 correlated with the expression of the BCL-6 (r=0.282, P<0.05), especially in the GCB-DLBCL (r=0.549, P<0.05). PFS was not significantly different between two groups of HBV (P>0.05), while the PFS in the R-CHOP regimen group was higher than that in the CHOP regimen group (P<0.05). COX multivariate analysis showed that both the chemotherapy regimen without R and the increased level of LDH were the risk factors affecting the prognosis of DLBCL patients (P<0.05).@*CONCLUSION@#EZH2 highly expresses in HBV positive group, suggesting that the significance of EZH2 in DLBCL with HBV infection is worth further explore.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Cyclophosphamide , Doxorubicin , Enhancer of Zeste Homolog 2 Protein , Genetics , Hepatitis B , Hepatitis B virus , Humans , Lymphoma, Large B-Cell, Diffuse , Genetics , Prognosis , Retrospective Studies , Rituximab , Vincristine
5.
Journal of Experimental Hematology ; (6): 2097-2012, 2020.
Article in Chinese | WPRIM | ID: wpr-880022

ABSTRACT

Enhancer of zeste homolog 2(EZH2) is a histone methyltransferase which regulate gene expression through epigenetic machinery. The abnormal expression of EZH2 has been described in many cancer types. With in-depth study, it was found that EZH2 is involved in the occurrence and development in many kinds of malignant hematologic disease which may play a dual role of oncogenes and tumor suppressor genes. In recent years, the emergence of EZH2 inhibitors provide a new option for the future treatment of hematological malignancies. In this review, the expression and clinical significance of EZH2 in various of hematological tumors were summarized briefly.


Subject(s)
Enhancer of Zeste Homolog 2 Protein/genetics , Hematologic Neoplasms/genetics , Humans , Neoplasms , Oncogenes , Research
6.
São Paulo; s.n; s.n; 2019. 122 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-1049921

ABSTRACT

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


Subject(s)
Plasmacytoma , RNA, Long Noncoding/adverse effects , Enhancer of Zeste Homolog 2 Protein/analysis , Androgens/analysis , Prostatic Neoplasms/diagnosis
7.
Journal of Experimental Hematology ; (6): 1402-1408, 2019.
Article in Chinese | WPRIM | ID: wpr-775707

ABSTRACT

OBJECTIVE@#To investigate the mechanism of rapamycin-induced apoptosis of chronic myelogenous leukemia cells.@*METHODS@#The chronic granulocytic leukemia K562 cells were divided into 3 groups: A, B and C group were treated with rapamycin of 10, 15 and 20 nmol/L, repectively for 24 h, while the K562 cells in control group were not treated with rapamycin. The effect of rapamycin on the proliferation of K562 cells was detected by MTT, and the effect of rapamycin on the apoptosis of K562 cells was detected by AnnexinV-FITC/PI double staining. The expression level of EZH2/Hedgehog signaling pathway genes in K562 cells was detected by RT-PCR, and Western blot was used to detect the levels of apoptotic protein and the related signaling pathway proteins in K562 cells.@*RESULTS@#The MTT assay showed that the different concentration of rapamycin had obvious inhibitory effects on the cells, and the survival rate of cells in group C was 37.6%±3.4%, which was significantly lower than that of the other groups (P<0.05). The apoptosis rate of cells in group C was 93.1%±8.1%, which was significantly higher than that of the other groups (P<0.05). By Western blot, it was found that the relative expression levels of Caspase-3 and BAX protein in group C were 0.36 ± 0.04 and 0.39±0.06, respectively, which were significantly higher than those in other groups (P<0.05), and the level of BCL-2 protein was 0.17±0.03, which was significantly lower than that of other groups (P<0.05). By RT-PCR, it was found that the mRNA levels of EZH2 and Hedgehog genes in A, B and C groups were significantly lower than those in the control group (P<0.05), but mRNA level of Ptch1 gene was significantly higher than that of the control (P<0.05). By Western blot, it was found that the expression levels of EZH2 and Hedgehog protein in A, B and C groups were significantly lower than that in the control group (P<0.05), but the level of Ptch1 protein was higher than that of the control (P<0.05). The relative levels of EZH2 and Hedgehog protein in group C were 0.21 ±0.03 and 0.16±0.05 respectively, which were significantly lower than those in other groups (P<0.05), and Ptch1 protein level were 0.46 ±0.06, significantly higher than that of other groups (P<0.05).@*CONCLUSION@#Rapamycin can inhibit the protein expression of EZH2 in leukemic cells, thus interfere with the activation of Hedgehog signaling pathway, promote the expression of apoptotic protein, reduce the level of anti apoptotic protein, and eventually induce apoptosis of leukemia cells.


Subject(s)
Apoptosis , Cell Proliferation , Enhancer of Zeste Homolog 2 Protein , Hedgehog Proteins , Humans , K562 Cells , Signal Transduction , Sirolimus
8.
Chinese Journal of Lung Cancer ; (12): 255-263, 2019.
Article in Chinese | WPRIM | ID: wpr-775634

ABSTRACT

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.


Subject(s)
Antineoplastic Agents , Pharmacology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Survival , Drug Resistance, Neoplasm , Drug Synergism , Enhancer of Zeste Homolog 2 Protein , ErbB Receptors , Gefitinib , Pharmacology , Humans , Lung Neoplasms , Pathology , Protein Kinase Inhibitors , Pharmacology
9.
Article in Chinese | WPRIM | ID: wpr-775061

ABSTRACT

OBJECTIVE@#To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR.@*METHODS@#The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection.@*RESULTS@#Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05).@*CONCLUSIONS@#Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.


Subject(s)
Child , Colon , Enhancer of Zeste Homolog 2 Protein , Genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors , Genetics , Hirschsprung Disease , Genetics , Humans , Infant, Newborn , RNA, Messenger
10.
Article in Chinese | WPRIM | ID: wpr-771878

ABSTRACT

OBJECTIVE@#To investigate the expression of miR-101 and EZH2 in patients with mantle cell lymphoma(MCL) and to analyze its correlation with clinical prognosis of MCL patients.@*METHODS@#RQ-PCR and S-P immunohistochemistry were used to detect the expressions of miR-101 and EZH2 in tissue of MCL patients. CCK-8 was used to assay the effect of miR-100 minics on the proliferation of Jeko-1 and Mino cells; the flow cytometry with Annexin V/PI double staining was used to assay the apoptosis; Western blot was used to assay the effect of miR-101 minics on the expression of EZH2 protein in Jeko-1 and Mino cells.@*RESULTS@#Compared with control group, miR-101 lowly expressed, and EZH2 protein highly expressed in MCL group, with very statistically significant difference(P<0.01).There was negative correlation between miR-101 and EZH2 expression(r=-0.638,P<0.05). The expression of miR-101 and EZH2 significantly correlated with B symptoms, International Prognostic Index(IPI) and Ann Arbor stage, respectively. Survival analysis showed that the overall survival(OS) rate of patients with low expression of miR-101 were significantly lower than that of patients with high miR-101 expression (P=0.0014), the OS rate of patients with EZH2 high expression were significantly lower than that of patients with EZH2 low expression (P=0.0093). The miR-100 minics could inhibit the proliferation of Jeko-1 and Mino cells, and increase the apoptotic rate. The expression of EZH2 protein was markedly suppressed by the miR-100 minics.@*CONCLUSION@#The expression of miR-101 and EZH2 is different in MCL patients with different clinical stage and prognosis. The miR-101 can inhibit the cell proliferation and induce cell apoptosis of mantle cell lymphoma by targeting EZH2.


Subject(s)
Apoptosis , Cell Proliferation , Enhancer of Zeste Homolog 2 Protein , Genetics , Humans , Lymphoma, Mantle-Cell , Genetics , MicroRNAs , Genetics , Prognosis
11.
Article in English | WPRIM | ID: wpr-772943

ABSTRACT

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.


Subject(s)
Adenosine , Metabolism , Adult Stem Cells , Cell Biology , Metabolism , Animals , Brain , Metabolism , Cell Differentiation , Genetics , Cell Proliferation , Enhancer of Zeste Homolog 2 Protein , Metabolism , Gene Expression Regulation , Methyltransferases , Metabolism , Mice, Inbred C57BL , Neural Stem Cells , Cell Biology , Metabolism , Neurogenesis , Genetics , Neurons , Cell Biology , Metabolism , RNA, Messenger , Genetics , Metabolism
12.
Chinese Journal of Lung Cancer ; (12): 358-364, 2018.
Article in Chinese | WPRIM | ID: wpr-776309

ABSTRACT

BACKGROUND@#It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology.@*METHODS@#In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR.@*RESULTS@#The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue.@*CONCLUSIONS@#The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.


Subject(s)
Animals , CRISPR-Cas Systems , Enhancer of Zeste Homolog 2 Protein , Genetics , Metabolism , Female , Gene Knockout Techniques , Gene Targeting , Humans , Lung Neoplasms , Genetics , Metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Guide
13.
Article in Chinese | WPRIM | ID: wpr-357524

ABSTRACT

<p><b>OBJECTIVE</b>To explore the therapeutic effect of enhancer of Zeste homolog 2 (EZH2) inhibitor GSK343 on periodontitis by regulating microphage differentiation.</p><p><b>METHODS</b>Macrophage RAW264.7 cells were divided into the blank (A group), control (B group), lipopolysaccharide (LPS) stimulation (C group), and LPS+GSK343 (D group) groups. Phenotype transformations was determined through Western blot analysis and enzyme-linked immunosorbent assay by detecting the differentiation of phenotypic biological markers, including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10), and Arginase-1 (Arg-1). Metergasis was identified by performing a phagocytosis test on Escherichia coli (E. coli).</p><p><b>RESULTS</b>Macrophage RAW264.7 cells produced classical phenotypic biomarkers (M1) TNF-α and iNOS under LPS stimulation. The expression levels of IL-10 and Arg-1 increased after adding GSK343 into the culture medium. GSK343 also induced the conversion of M1 macrophages into M2 macrophages. Macrophage RAW264.7 cells exerted a phagocytic effect on E. coli, and this effect was enhanced after adding LPS into the culture medium. GSK343 regulated the macrophage RAW264.7 phagocytosis of E. coli.</p><p><b>CONCLUSIONS</b>GSK343 possibly participates in the regulation of macrophage differentiation and, consequently, in the latent treatment of periodontitis.</p>


Subject(s)
Arginase , Cell Differentiation , Enhancer of Zeste Homolog 2 Protein , Enzyme Inhibitors , Pharmacology , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Indazoles , Pharmacology , Interleukin-10 , Lipopolysaccharides , Macrophages , Nitric Oxide Synthase Type II , Periodontitis , Phagocytosis , Pyridones , Pharmacology , Tumor Necrosis Factor-alpha
14.
Rev. bras. ginecol. obstet ; 38(6): 280-286, June 2016. tab, graf
Article in English | LILACS | ID: lil-789043

ABSTRACT

Abstract Introduction Neoadjuvant chemotherapy (NAC) is the standard treatment for locally advanced breast cancer. However, some tumors will not respond to this treatment due to histological and molecular features. The protein EZH2 (enhancer of zest homolog 2) is a histone methyltransferase that is correlated with poorly differentiated breast carcinomas and aggressive tumor behavior. Purpose The present study evaluated the association between EZH2 expression and response to NAC, and its correlation with HER2 overexpression, estrogen and progesterone receptors (ER, PR) and Ki-67 proliferation index. Methods A total of 60 patients with locally advanced breast cancer treated with NAC were selected for this study. Twenty-three paraffin blocks had not enough material for tissue resection, and were not evaluated. A tissue microarray based in immunohistochemistry (IHC) analysis of EZH2 was performed for the remaining 37 specimens. Patients were divided into two groups based on response to NAC. Results EZH2 expression was significantly associated with markers of poor prognosis such as ER negativity (p = 0.001), PR negativity (p = 0.042) and high Ki-67 proliferation index (p = 0.002). High EZH2 expression was not correlated with the response to NAC. Conclusions Our data suggested that EZH2 protein expression may not correlate with the clinical response to NAC. Other studies with more patients are needed to confirm this observation.


Resumo Introdução A quimioterapia neoadjuvante é o tratamento padrão para os cânceres de mama localmente avançados. Entretanto, apenas uma porcentagem desses tumores irá responder ao tratamento, devido a características histológicas e moleculares. A proteína EZH2 (enhancer of zest homolog 2) é uma histona metiltransferase associada a tumores mal diferenciados e de comportamento agressivo. Objetivo O presente estudo teve como objetivo avaliar a associação entre a expressão da proteína EZH2 e a resposta à quimioterapia neoadjuvante, além da correlação dessa proteína com hiper-expressão de HER2, receptores de estrogênio e progesterona, e o marcador de proliferação Ki-67. Métodos Um total de 60 pacientes com câncer de mama localmente avançado tratadas com quimioterapia neoadjuvante foram selecionadas para esse estudo. Vinte e três blocos de parafina não continham material suficiente para ressecção e não foram avaliados. Foi realizado microarray baseado em análise imuno-histoquímica da proteína EZH2 para as 36 pacientes restantes. As pacientes foram divididas em dois grupos baseado na resposta à quimioterapia neoadjuvante. Resultados A expressão da proteína EZH2 foi significativamente associada com marcadores de pior prognóstico, como negatividade para receptor de estrogênio (p = 0,001) e progesterona (p = 0,042), além de alto Ki-67 (p = 0,002). Entretanto, a alta expressão da EZH2 não se correlacionou com a resposta à quimioterapia neoadjuvante. Conclusões Nossos dados sugerem que a expressão da proteína EZH2 pode não estar relacionada com a resposta clínica à quimioterapia neoadjuvante. Outros estudos com maior número de pacientes são necessários para confirmar esses achados.


Subject(s)
Humans , Female , Middle Aged , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Breast Neoplasms/pathology , Case-Control Studies , Chemotherapy, Adjuvant , Gene Expression Regulation, Neoplastic , Neoadjuvant Therapy , Neoplasm Staging , Pilot Projects , Treatment Outcome
15.
Article in Chinese | WPRIM | ID: wpr-239578

ABSTRACT

To investigate the effect of a novel EZH2 inhibitor GSK126 on cell growth, apoptosis and migration of prostate cancer cells.Prostate cancer PC-3 and DU145 cells were treated with GSK126 at different doses. Cell growth was detected by sulforhodamine assay. Cell apoptosis was assayed by Annexin V-/PI kit. Transwell chamber and wound healing assays were conducted to detect cell migration. The mRNA level was detected by quantitative PCR, and protein expression was detected by Western blot analysis.GSK126 showed significant effect on cell growth and apoptosis when the dose was higher than 50 μmol/L. Wound healing assay revealed that scratch space in PC-3 cells was significantly increased in a dose-dependent manner in GSK126-treated groups[(247.2±24.4),(347.2±19.2) and (410.5±18.1) μm in low, medium and high dose (5.0, 20.0, 50.0 μmol/L), respectively] as compared with the control group[(171.3±17.8) μm](all<0.05). Transwell assay showed that migrated PC-3 cells in control group was 322.0±17.9,while those in GSK126-treated groups were 198.3±15.4 (low),82.7±6.2 (medium) and 30.2±4.1 (high), and the differences between the control group and GSK126-treated groups were significant(all<0.05). In addition, GSK126 up-regulated E-cadherin mRNA expression and down-regulated N-cadherin and Vimentin mRNA expression, whereas had no significant effect on Snail, Fibronectin and VEGF-A mRNA expression. The protein expression of E-cadherin was elevated but VEGF-A protein did not change in GSK126-treated groups. Similar results were exhibited in DU145 cell.GSK126 can significantly inhibit cell migration and invasion in prostate cancer PC-3 and DU145 cells, which may be resulted from its effect on epithelial-mesenchymal transition. GSK126 may be used as a potential anti-prostate cancer dug in clinic.


Subject(s)
Apoptosis , Cadherins , Metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Down-Regulation , Drug Screening Assays, Antitumor , Methods , Enhancer of Zeste Homolog 2 Protein , Metabolism , Fibronectins , Metabolism , Humans , Indoles , Pharmacology , Male , Prostatic Neoplasms , Chemistry , Genetics , Pyridones , Pharmacology , RNA, Messenger , Up-Regulation , Vascular Endothelial Growth Factor A , Vimentin , Metabolism
16.
Article in Chinese | WPRIM | ID: wpr-239238

ABSTRACT

<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>


Subject(s)
Carcinoma, Squamous Cell , Diagnosis , Metabolism , Enhancer of Zeste Homolog 2 Protein , Esophageal Neoplasms , Diagnosis , Metabolism , Humans , Lymphatic Metastasis , Polycomb Repressive Complex 2 , Metabolism , Postoperative Period , Prognosis
17.
National Journal of Andrology ; (12): 500-503, 2015.
Article in Chinese | WPRIM | ID: wpr-276069

ABSTRACT

<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>


Subject(s)
Androgens , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein , Genetic Vectors , Humans , Male , MicroRNAs , Physiology , Polycomb Repressive Complex 2 , Genetics , Metabolism , Prostatic Neoplasms , Metabolism , RNA, Messenger , Metabolism , Transfection
18.
Protein & Cell ; (12): 265-296, 2014.
Article in English | WPRIM | ID: wpr-757506

ABSTRACT

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.


Subject(s)
Chromatin , Metabolism , Chromatin Assembly and Disassembly , DNA Methylation , Enhancer of Zeste Homolog 2 Protein , Epigenesis, Genetic , Histones , Metabolism , Humans , Neoplasms , Genetics , Metabolism , Pathology , Polycomb Repressive Complex 2 , Genetics , Metabolism
19.
Article in Chinese | WPRIM | ID: wpr-356919

ABSTRACT

<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>


Subject(s)
3' Untranslated Regions , Breast Neoplasms , Genetics , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Profiling , Humans , Lentivirus , Genetics , MicroRNAs , Genetics , Polycomb Repressive Complex 2 , Genetics
20.
Chinese Medical Journal ; (24): 1077-1083, 2014.
Article in English | WPRIM | ID: wpr-253195

ABSTRACT

<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>


Subject(s)
Breast Neoplasms , Metabolism , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Histones , Metabolism , Humans , MCF-7 Cells , Polycomb Repressive Complex 2 , Metabolism , Receptors, Prolactin , Metabolism , Tumor Suppressor Protein p53 , Metabolism
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