RESUMO
Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.
Assuntos
Neoplasias Encefálicas/terapia , Ciclina D2/biossíntese , Metilases de Modificação do DNA/biossíntese , Enzimas Reparadoras do DNA/biossíntese , Glioblastoma/terapia , Magnetoterapia , Temozolomida/farmacologia , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Ciclina D2/genética , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Campos Eletromagnéticos , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. METHODS: Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. RESULTS: We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. CONCLUSIONS: Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.
Assuntos
Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Mieloma Múltiplo/genética , Aneuploidia , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/ultraestrutura , Ciclina D1/biossíntese , Ciclina D1/genética , Ciclina D2/biossíntese , Ciclina D2/genética , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Código das Histonas , Histonas/metabolismo , Humanos , Mieloma Múltiplo/classificação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Plasmócitos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-maf/genética , Translocação GenéticaRESUMO
Bisphenol A (BPA), as synthetic monomer used in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Epigenetic alterations could act an important role in BPA-induced toxicity, but its mechanism has not been fully understood. We investigated the effects of BPA on gene expression of chromatin modifying enzymes, promoter methylation of tumor suppressor genes and histone modifications in human prostate carcinoma cells (PC-3). IC50 value of BPA was determined as 217 and 190⯵M in PC-3â¯cells by MTT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10⯵M of BPA for 96â¯h. We observed a significant increase on promoter DNA methylation and decrease on gene expression of p16 gene while no change was observed for Cyclin D2 and Rassf1. Significant changes were observed in global histone modifications (H3K9ac, H3K9me3, H3K27me3, and H4K20me3) in PC-3â¯cells. According to these results, we investigated wide-range epigenetic modifications using PCR arrays. After 96â¯h BPA exposure, chromatin modifying enzymes including KDM5B and NSD1 were significantly downregulated. Also, promoter methylation of tumor suppressor genes including BCR, GSTP1, LOX, MGMT, NEUROG1, PDLIM4, PTGS2, PYCARD, TIMP3, TSC2 and ZMYDN10 altered significantly. ChIP results showed that H3K9ac, H3K9me3 and H3K27me3 modifications on p16 gene showed significant increases after 1 and 10⯵M of BPA exposure. In conclusion, epigenetic signatures such as DNA methylation and histone modifications could be proposed as molecular biomarkers of BPA-induced prostate cancer progression.
Assuntos
Compostos Benzidrílicos/toxicidade , Metilação de DNA/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Código das Histonas/efeitos dos fármacos , Fenóis/toxicidade , Neoplasias da Próstata/induzido quimicamente , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Ciclina D2/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Inibidor Tecidual de Metaloproteinase-3 , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Grx3; Glrx3)) is a ubiquitous protein that can interact with the embryonic ectoderm development (EED) protein via each of its two C-terminal PICOT/Grx homology domains. Since EED is a Polycomb-Group protein and a core component of the polycomb repressive complex 2 (PRC2), we tested the involvement of PICOT in the regulation of PRC2-mediated H3 lysine 27 trimethylation (H3K27me3), transcription and translation of selected PRC2 target genes. A fraction of the cellular PICOT protein was found in the nuclei of leukemia cell lines, where it was associated with the chromatin. In addition, PICOT coimmunoprecipitated with chromatin-residing EED derived from Jurkat and COS-7 cell nuclei. PICOT knockdown led to a reduced H3K27me3 mark and a decrease in EED and EZH2 at the CCND2 gene promoter. In agreement, PICOT-deficient T cells exhibited a significant increase in CCND2 mRNA and protein expression. Since elevated expression levels of PICOT were reported in several different tumors and correlated in the current studies with decreased transcription and translation of the CCND2 gene, we tested whether this opposite correlation exists in human cancers. Data from the Cancer Genome Atlas (TCGA) database indicated statistically significant negative correlation between PICOT and CCND2 in eight different human tumors where the highest correlation was in lung (p = 8.67E-10) and pancreatic (p = 1.06E-5) adenocarcinoma. Furthermore, high expression of PICOT and low expression of CCND2 correlated with poor patient survival in five different types of human tumors. The results suggest that PICOT binding to chromatin-associated EED modulates the H3K27me3 level at the CCND2 gene promoter which may be one of the potential mechanisms for regulation of cyclin D2 expression in tumors. These findings also indicate that a low PICOT/CCND2 expression ratio might serve as a good predictor of patient survival in selected human cancers.
Assuntos
Proteínas de Transporte/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Ciclina D2/biossíntese , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Células A549 , Animais , Células COS , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Ciclina D2/genética , Células HEK293 , Histonas/genética , Humanos , Células Jurkat , Células K562 , Complexo Repressor Polycomb 2/genética , Regiões Promotoras Genéticas , Células THP-1 , Células U937RESUMO
The present study aimed to explore the effect of the long noncoding RNA TP73 antisense RNA 1 (TP73AS1) on cervical cancer progression. Cervical cancer and adjacent tissues were collected from 56 patients and assessed. In addition, HeLa and CaSki cells were transfected with various plasmids, inhibitors and corresponding controls, and then Cell Counting Kit8 and Transwell assays were used to detect the cell proliferation, migration and invasion abilities. Luciferase reporter gene assay was also performed in HeLa cells. Reverse transcriptionquantitative polymerase chain reaction (RTqPCR) was used to investigate TP73AS1, microRNA607 (miR607) and cyclin D2 (CCND2) gene expression, while CCND2 protein expression was determined by western blot analysis. The results revealed that the TP73AS1 level was upregulated in cervical cancer tissues (P<0.05) and predicted a poor 5year overall survival (P<0.05). HeLa and CaSki cells transfected with siTP73AS1 exhibited reduced proliferation, migration and invasion abilities when compared with those in the siNC group (P<0.05). Furthermore, miR607 was found to be negatively regulated by TP73AS1, while CCND2 was negatively regulated by miR607. HeLa and CaSki cells transfected with siTP73AS1 exhibited lower CCND2 mRNA and protein expression levels compared with the siNC and siTP73AS1 + miRinhibitor groups (P<0.05). Compared with the siNC and siTP73AS1 + CCND2 overexpression groups, siTP73AS1transfected HeLa and CaSki cells had decreased proliferation, migration and invasion abilities (P<0.05). In conclusion, the findings suggested that upregulation of TP73AS1 promoted cervical cancer progression by promoting CCND2 via the suppression of miR607 expression.
Assuntos
Movimento Celular , Proliferação de Células , Ciclina D2/biossíntese , Regulação Neoplásica da Expressão Gênica , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Neoplasias do Colo do Útero/metabolismo , Feminino , Células HeLa , Humanos , Neoplasias do Colo do Útero/patologiaRESUMO
MicroRNAs (miRNAs/miRs) serve important roles in the chemotherapeutic effect of anticancer drugs. To investigate the roles of miRNAs in cisplatininduced suppression of lung adenocarcinoma cell proliferation, A549 cells were treated with different concentrations of cisplatin. An MTT assay demonstrated that cisplatin inhibited A549 cell proliferation in a dosedependent manner. Cisplatin induced cell apoptosis and inhibited cell migration by increasing the levels of miR93, miR26a and miR26b. Furthermore, as an upstream factor, miR93 was proposed to regulate cyclin D2 expression in miR93transfected A549 cells. Cisplatin also induced Bcl2associated X protein expression, and decreased that of Bcl2 and cMyc in lung adenocarcinoma cells. In vivo analysis further supported that cisplatin inhibited lung adenocarcinoma cell growth by regulating cyclin D2 and miR93 expression. In conclusion, our findings demonstrated that cisplatin could effectively inhibit lung adenocarcinoma cell proliferation by decreasing cyclin D2 expression via miR93.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Cisplatino/farmacologia , Ciclina D2/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Regulação para Cima/efeitos dos fármacos , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologiaRESUMO
OBJECTIVE: This study aimed to detect the expression of miR-373-3p and CCND2 in gemcitabine-resistance pancreatic carcinoma (PC) cells, investigate the relationship between miR-373-3p and CCND2, and explore their effects on PC propagation, migration, invasion and apoptosis. METHODS: R software was applied for analyzing differentially expressed genes (DEGs) in cell samples. The potential biological pathway was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, based on R software. The gemcitabine-resistance PC cells were screened out using MTT assay, and they were applied in the next experiments. MiR-373-3p and CCND2 expression in GEM-PANC-1 cells were measured by qRT-PCR. After transfection, the expression of CCND2 protein was examined via western blot assay. Cells viability and apoptosis were confirmed by MTT proliferation assay and Flow cytometry, whereas cells migration and invasion were analyzed by transwell assay. The targeting relationship between miR-373-3p and CCND2 was identified by dual-luciferase reporter assay. RESULTS: MiR-373-3p was found to be low expressed in GEM-PANC-1 cells while CCND2 was highly expressed in GEM-PANC-1 cells. MiR-373-3p negatively regulated CCND2 expression through KEGG_Cell_Cycle_Signaling_Pathway. The targeted relationship between miR-373-3p and CCND2 could be verified using dual luciferase reporter assay. MTT proliferation assay, transwell assay and Annexin V assay demonstrated that miR-373-3p suppressed GEM-PANC-1 cells propagation and invasion and promoted cell apoptosis, while CCND2 showed totally reverse effects compared with miR-373-3p. All the results suggested that miR-373-3p could enhance the chemosensitivity of GEM-PANC-1 cells by regulating CCND2. CONCLUSION: MiR-373-3p inhibited cell propagation, migration and invasion and boosted apoptosis in gemcitabine resistance pancreatic carcinoma cells by targeting CCND2.
Assuntos
Ciclina D2/biossíntese , Desoxicitidina/análogos & derivados , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MicroRNAs/biossíntese , Neoplasias Pancreáticas/metabolismo , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular , Linhagem Celular Tumoral , Ciclina D2/antagonistas & inibidores , Desoxicitidina/administração & dosagem , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Neoplasias Pancreáticas/patologia , Gencitabina , Neoplasias PancreáticasRESUMO
Given the frequent and largely incurable occurrence of multiple myeloma, identification of germline genetic mutations that predispose cells to multiple myeloma may provide insight into disease etiology and the developmental mechanisms of its cell of origin, the plasma cell (PC). Here, we identified familial and early-onset multiple myeloma kindreds with truncating mutations in lysine-specific demethylase 1 (LSD1/KDM1A), an epigenetic transcriptional repressor that primarily demethylates histone H3 on lysine 4 and regulates hematopoietic stem cell self-renewal. In addition, we found higher rates of germline truncating and predicted deleterious missense KDM1A mutations in patients with multiple myeloma unselected for family history compared with controls. Both monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma cells have significantly lower KDM1A transcript levels compared with normal PCs. Transcriptome analysis of multiple myeloma cells from KDM1A mutation carriers shows enrichment of pathways and MYC target genes previously associated with myeloma pathogenesis. In mice, antigen challenge followed by pharmacologic inhibition of KDM1A promoted PC expansion, enhanced secondary immune response, elicited appearance of serum paraprotein, and mediated upregulation of MYC transcriptional targets. These changes are consistent with the development of MGUS. Collectively, our findings show that KDM1A is the first autosomal-dominant multiple myeloma germline predisposition gene providing new insights into its mechanistic roles as a tumor suppressor during post-germinal center B-cell differentiation.Significance: KDM1A is the first germline autosomal dominant predisposition gene identified in multiple myeloma and provides new insights into multiple myeloma etiology and the mechanistic role of KDM1A as a tumor suppressor during post-germinal center B-cell differentiation. Cancer Res; 78(10); 2747-59. ©2018 AACR.
Assuntos
Predisposição Genética para Doença/genética , Histona Desmetilases/genética , Gamopatia Monoclonal de Significância Indeterminada/genética , Mieloma Múltiplo/genética , Animais , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Genes Supressores de Tumor , Células Germinativas/patologia , Histona Desmetilases/antagonistas & inibidores , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto/genética , Paraproteínas/análise , Plasmócitos/patologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
RATIONALE: The effectiveness of transplanted, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for treatment of ischemic myocardial injury is limited by the exceptionally low engraftment rate. OBJECTIVE: To determine whether overexpression of the cell cycle activator CCND2 (cyclin D2) in hiPSC-CMs can increase the graft size and improve myocardial recovery in a mouse model of myocardial infarction by increasing the proliferation of grafted cells. METHODS AND RESULTS: Human CCND2 was delivered to hiPSCs via lentiviral-mediated gene transfection. In cultured cells, markers for cell cycle activation and proliferation were ≈3- to 7-folds higher in CCND2-overexpressing hiPSC-CMs (hiPSC-CCND2OECMs) than in hiPSC-CMs with normal levels of CCND2 (hiPSC-CCND2WTCMs; P<0.01). The pluripotent genes (Oct 4, Sox2, and Nanog) decrease to minimal levels and undetectable levels at day 1 and 10 after differentiating to CMs. In the mouse myocardial infarction model, cardiac function, infarct size, and the number of engrafted cells were similar at week 1 after treatment with hiPSC-CCND2OECMs or hiPSC-CCND2WTCMs but was about tripled in hiPSC-CCND2OECM-treated than in hiPSC-CCND2WTCM-treated animals at week 4 (P<0.01). The cardiac function and infarct size were significantly better in both cell treatment groups' hearts than in control hearts, which was most prominent in hiPSC-CCND2OECM-treated animals (P<0.05, each). No tumor formation was observed in any hearts. CONCLUSIONS: CCND2 overexpression activates cell cycle progression in hiPSC-CMs that results in a significant enhanced potency for myocardial repair as evidenced by remuscularization of injured myocardium. This left ventricular muscle regeneration and increased angiogenesis in border zone are accompanied by a significant improvement of left ventricular chamber function.
Assuntos
Ciclina D2/biossíntese , Ventrículos do Coração/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Regeneração/fisiologia , Animais , Células Cultivadas , Ciclina D2/genética , Expressão Gênica , Células HEK293 , Ventrículos do Coração/lesões , Ventrículos do Coração/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/terapia , Miócitos Cardíacos/transplanteRESUMO
The objective of this study was to validate the diagnostic and clinical role of four protein products of genes previously found to be differentially expressed in uterine low-grade endometrial stromal sarcoma (LG-ESS) compared to uterine leiomyosarcoma (LMS). Protein expression by immunohistochemistry of transgelin (TGLN), neuron navigator-2 (NAV2), fatty acid binding protein-3 (FABP3), and cyclin D2 (CCND2) was analyzed in 305 uterine sarcomas (231 LMS, 74 LG-ESS). Expression was analyzed for association with clinicopathologic parameters and survival. TGLN (p < 0.001), NAV2 (p < 0.001), and FABP3 (p = 0.005) were overexpressed in LMS compared to LG-ESS, whereas nuclear CCND2 (p < 0.001) was overexpressed in LG-ESS. NAV2 expression was associated with shorter overall survival in patients with LMS (p = 0.037), whereas nuclear CCND2 expression in LG-ESS was significantly related to longer survival (p = 0.012) in univariate analysis. Nuclear CCND2 expression was an independent prognosticator in Cox multivariate analysis (p = 0.023). In conclusion, TGLN, FABP3, NAV2, and nuclear CCND2 aid in differentiating LG-ESS from LMS. NAV2 and CCND2 are novel candidate prognostic markers in LMS and LG-ESS, respectively.
Assuntos
Biomarcadores Tumorais/análise , Ciclina D2/biossíntese , Leiomiossarcoma/diagnóstico , Proteínas do Tecido Nervoso/biossíntese , Sarcoma do Estroma Endometrial/diagnóstico , Neoplasias Uterinas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Helicases , Diagnóstico Diferencial , Feminino , Humanos , Estimativa de Kaplan-Meier , Leiomiossarcoma/mortalidade , Leiomiossarcoma/patologia , Pessoa de Meia-Idade , Prognóstico , Sarcoma do Estroma Endometrial/mortalidade , Sarcoma do Estroma Endometrial/patologia , Neoplasias Uterinas/mortalidade , Neoplasias Uterinas/patologia , Adulto JovemRESUMO
OBJECTIVE: To determine the differential protein expression of biomarkers FGFR3, PI3K (subunits PI3Kp110α, PI3KClassIII, PI3Kp85), AKT, p21Waf1/Cip1 and cyclins D1 and D3 in T1 bladder cancer versus healthy tissue and to study their potential role as early recurrence markers. MATERIAL AND METHOD: This is a prospective study that employed a total of 67 tissue samples (55 cases of T1 bladder tumours that underwent transurethral resection and 12 cases of adjacent healthy mucosa). The protein expression levels were assessed using Western blot, and the means and percentages were compared using Student's t-test and the chi-squared test. The survival analysis was conducted using the Kaplan-Meier method and the log-rank test. RESULTS: Greater protein expression was detected for FGFR3, PI3Kp110α, PI3KClassIII, cyclins D1 and D3 and p21Waf1/Cip1 in the tumour tissue than in the healthy mucosa. However, these differences were not significant for PI3Kp85 and AKT. We observed statistically significant correlations between early recurrence and PI3Kp110α, PI3KClassIII, PI3Kp85 and AKT (P=.003, P=.045, P=.050 and P=.028, respectively), between the tumour type (primary vs. recurrence) and cyclin D3 (P=.001), between the tumour size and FGFR3 (P=.035) and between multifocality and cyclin D1 (P=.039). The survival analysis selected FGFR3 (P=.024), PI3Kp110α (P=.014), PI3KClassIII (P=.042) and AKT (P=.008) as markers of early-recurrence-free survival. CONCLUSIONS: There is an increase in protein expression levels in bladder tumour tissue. The overexpression of FGFR3, PI3Kp110α, PI3KClassIII and AKT is associated with increased early-recurrence-free survival for patients with T1 bladder tumours.
Assuntos
Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Idoso , Idoso de 80 Anos ou mais , Ciclina D1/biossíntese , Ciclina D2/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteína Oncogênica v-akt/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Prognóstico , Estudos Prospectivos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Análise de Sobrevida , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
AIM: This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. METHODS: The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. RESULTS: Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. CONCLUSION: We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle.
Assuntos
Ciclina D2/biossíntese , Endométrio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/metabolismo , Progesterona/farmacologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchoragedependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Ciclina D1/biossíntese , Ciclina D2/biossíntese , Neoplasias Bucais/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/biossíntese , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Caderinas/biossíntese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D2/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/biossíntese , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lapatinib , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Quinazolinas/administração & dosagem , Receptor ErbB-2/genética , Receptor ErbB-3/genéticaRESUMO
Data derived from genomic and transcriptomic analyses have revealed that long noncoding RNAs (lncRNAs) have important roles in the transcriptional regulation of various genes. Recent studies have identified the mechanism underlying this function. To date, a variety of noncoding transcripts have been reported to function in conjunction with epigenetic regulator proteins. In this study, we investigated the function of linc00598, which is transcribed by a genomic sequence on chromosome 13, downstream of FoxO1 and upstream of COG6. Microarray analysis showed that linc00598 regulates the transcription of specific target genes, including those for cell cycle regulators. We discovered that linc00598 regulates CCND2 transcription through modulation of the transcriptional regulatory effect of FoxO1 on the CCND2 promoter. Moreover, we observed that knockdown of linc00598 induced G0/G1 cell cycle arrest and inhibited proliferation. These data indicate that linc00598 plays an important role in cell cycle regulation and proliferation through its ability to regulate the transcription of CCND2.
Assuntos
Ciclina D2/biossíntese , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , RNA Longo não Codificante/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Ciclina D2/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Humanos , Células K562 , Células MCF-7 , RNA Longo não Codificante/genética , Fase de Repouso do Ciclo Celular/fisiologia , Células THP-1RESUMO
Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a ß-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases ß-catenin occupancy on the MYC promoter, (2) forms a complex with ß-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM.
Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Mucina-1/biossíntese , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elementos de Resposta , Transcrição Gênica , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Ciclina D2/genética , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Humanos , Mucina-1/genética , Mieloma Múltiplo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/genética , Telomerase/biossíntese , Telomerase/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent.
Assuntos
Antineoplásicos/uso terapêutico , Hidrazonas/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Fator de Transcrição E2F1/biossíntese , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Nus , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/biossínteseRESUMO
The present study investigated the effects of microRNA-124-3p (miR-124-3p) expression on nasopharyngeal carcinoma (NPC) cells and its relevant mechanism. A total of 90 NPC tissues and 85 postnasal catarrh tissues were collected. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect tissue samples and expression of miR-124-3p in CNE1, CNE2, SUNE1, H0NE1, 5-8F, 6-10B and C666-1 NPC cell line and immortalized nasopharyngeal epithelial cells line (NP69). Overexpressed miRNA-124-3p in CNE-2 was downregulated, and low-expressed miRNA124-3p in C666-1 was upregulated by liposome-mediated transfection. Cell Counting Kit-8 (CCK-8), flow cytometry, the scratch test, Transwell migration assay and Boyden chamber assays were used to detect cell proliferation, apoptosis, migration and invasion. The target gene of miRNA-124-3 calculated by bioinformatics was further determined using dual-luciferase system. Protein levels of the signal transducers and activators of transcription 3 (STAT3), phospho-STAT3 (p-STAT3), mouse anti-human cyclin D2 (CCND2) and matrix metalloproteinase-2 (MMP-2) were tested by western blotting. miRNA-124-3p expression in NPC was markedly downregulated compared to postnasal catarrh tissues (P<0.001); miRNA-124-3p expression showed close linkage with clinical stages, regional lymph node involvement and T stages (all P<0.001). miRNA-124-3p expression was lower in the 7 NPC cell lines than NP69 cells (all P<0.05). After upregulation of miR-124-3p, proliferation, apoptosis, migration and invasion of C666-1 cells were suppressed; while after downregulation of miR-124-3p, CNE2 cells were increased (all P<0.05). Expression of STAT3, p-STAT3, CCND2 and MMP-2 in C666-1 cells was decreased after transfection with miRNA-124-3p, and the above protein expression in CNE-2 cells was increased after inhibition of miRNA-124-3p (all P<0.05). To sum up, this study shows that miR-124-3p may negatively regulate the transcription of the STAT3 by interfering with its 3'UTR, and the degradation of STAT3 affects its downstream expression of such as p-STAT3, CCND2 and MMP-2, thereby promoting NPC cells apoptosis and inhibiting proliferation, migration and invasion of NPC cells.
Assuntos
Ciclina D2/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , MicroRNAs/genética , Neoplasias Nasofaríngeas/genética , Fator de Transcrição STAT3/biossíntese , Regiões 3' não Traduzidas , Animais , Apoptose/genética , Carcinoma , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D2/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 2 da Matriz/genética , Camundongos , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Metástase Neoplásica , Fator de Transcrição STAT3/genéticaRESUMO
Ppard(-/-) mice exhibit smaller litter size compared with Ppard(+/+) mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard(-/-) mice compared with Ppard(+/+) mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard(+/+) mice as compared with Ppard(-/-) mice, and these were associated with decreased Sertoli cell number in Ppard(+/+) mice. Cyclin D1 and cyclin D2 expression was lower in Ppard(+/+) as compared with Ppard(-/-) mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard(+/+) mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.
Assuntos
Proliferação de Células/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Animais , Linhagem Celular , Claudinas/biossíntese , Claudinas/genética , Ciclina D1/biossíntese , Ciclina D1/genética , Ciclina D2/biossíntese , Ciclina D2/genética , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/genética , Masculino , Camundongos , Receptores Citoplasmáticos e Nucleares/genética , Células de Sertoli/citologiaRESUMO
Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.
Assuntos
Ciclina D2/genética , Metilação de DNA/genética , Neoplasias de Cabeça e Pescoço/genética , Tolerância a Radiação/genética , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Linhagem Celular Tumoral , Ilhas de CpG , Ciclina D2/biossíntese , Metilação de DNA/efeitos da radiação , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Fator 4 Semelhante a Kruppel , Regiões Promotoras GenéticasRESUMO
The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.