RESUMO
OBJECTIVE: Long Non-Coding RNAs (LncRNAs) act as an indispensable role in cancer development. The study aimed to investigate the role and mechanism of lncRNA Small Nucleolar RNA Host Gene 1 (SNHG1) in Bladder Cancer (BC) progression. METHOD: The expression, prognostic value, diagnostic value, and correlation of SNHG1, Enhancer of Zeste 2 polycomb repressive complex 2 subunit (EZH2), and Kruppel Like Factor 2 (KLF2) were analyzed through bioinformatics analysis. The expression was also validated in BC tissues and cell lines. Besides, their regulation and binding were tested via qPCR, Western blot, Dual-Luciferase Reporter Assay (DLRA), Argonaute RISC catalytic component 2-RNA Immunoprecipitation (AGO2-RIP), and Chromatin Immunoprecipitation (ChIP). A xenograft model in nude mice was also established. RESULTS: SNHG1 was significantly overexpressed in BC tissues and cells. Importantly, SNHG1 was associated with poor survival, and ROC curves revealed high diagnostic values. Moreover, by CCK8, wound healing, transwell, and Western blot analysis, SNHG1 knockdown significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of BC cells. Additionally, in vivo experiments showed that silencing SNHG1 hindered tumorigenesis and tumor growth. Regarding mechanism, the results of AGO2-RIP, ChIP or DLRA showed that SNHG1 played different roles at diverse subcellular sites. In the cytoplasm, SNHG1 acted as a competing endogenous RNA for miR-137-3p to promote EZH2 expression. In the nucleus, SNHG1 could interact with EZH2 to inhibit KLF2 transcription. CONCLUSION: Our study elucidated that SNHG1 formed a regulatory network and played an oncogenic role in BC, which provided a novel therapeutic target for BC treatment.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs , RNA Longo não Codificante , Neoplasias da Bexiga Urinária , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Nus , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias da Bexiga Urinária/genéticaRESUMO
Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glicocálix/metabolismo , Ácido Hialurônico/metabolismo , Sindecana-1/genética , Neoplasias de Mama Triplo Negativas/genética , Via de Sinalização Wnt/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Antígeno CD24/genética , Antígeno CD24/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Bases de Dados Factuais , Feminino , Glicocálix/química , Glicocálix/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/farmacologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Células MCF-7 , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sobrevida , Sindecana-1/antagonistas & inibidores , Sindecana-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Esclerose Lateral Amiotrófica/metabolismo , Brasil , Linhagem Celular , Humanos , Cariótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.
Assuntos
Encéfalo/fisiologia , Terapia Genética/métodos , Fatores de Transcrição Kruppel-Like/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Regeneração , Fatores de Transcrição SOXB1/genética , Adenoviridae/genética , Animais , Células Cultivadas , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOXB1/metabolismoRESUMO
OBJECTIVES: The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS: Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Fator 3 de Transcrição de Octâmero/fisiologia , Animais , Bovinos , Reprogramação Celular , Fibroblastos , Regulação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Lentivirus , Fatores de Transcrição SOXB1/metabolismo , SuínosRESUMO
Cervical cancer (CC) is associated with alterations in immune system balance, which is primarily due to a shift from Th1 to Th2 and the unbalance of Th17/Treg cells. Using in silico DNA copy number analysis, we have demonstrated that ~20% of CC samples exhibit gain of 8q22.3 and 19q13.31; the regions of the genome that encodes the KLF10 and PSG genes, respectively. Gene expression studies demonstrated that there were no alterations in KLF10 mRNA expression, whilst the PSG2 and -5 genes were up-regulated by 1.76 and 3.97-fold respectively in CC compared to normal tissue controls. siRNA and ChIP experiments in SiHa cells have demonstrated that KLF10 participates in immune response through regulation of IL6, IL25 and PSG2 and PSG5 genes. Using cervical tissues from KLF10-/- mice, we have identified down-regulation of PSG17, -21 and -23 and IL11. These results suggest that KLF10 may regulate immune system response genes in cervical cancer among other functions. KLF10 and PSG copy number variations and alterations in mRNA expression levels could represent novel molecular markers in CC.
Assuntos
Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Glicoproteínas beta 1 Específicas da Gravidez/genética , Neoplasias do Colo do Útero/genética , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Feminino , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Glicoproteínas beta 1 Específicas da Gravidez/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/imunologiaRESUMO
Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.
Assuntos
Proteínas de Peixes/metabolismo , Linguado/metabolismo , Músculo Esquelético/metabolismo , Estresse Psicológico/metabolismo , Fatores Etários , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Doença Crônica , Aglomeração , Modelos Animais de Doenças , Proteínas de Peixes/genética , Linguado/sangue , Linguado/genética , Linguado/crescimento & desenvolvimento , Regulação da Expressão Gênica , Hidrocortisona/sangue , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Estresse Psicológico/genética , Estresse Psicológico/fisiopatologiaRESUMO
Enhanced carotid body (CB) chemoreflex function is strongly related to cardiorespiratory disorders and disease progression in heart failure (HF). The mechanisms underlying CB sensitization during HF are not fully understood, however previous work indicates blood flow per se can affect CB function. Then, we hypothesized that the CB-mediated chemoreflex drive will be enhanced only in low output HF but not in high output HF. Myocardial infarcted rats and aorto-caval fistulated rats were used as a low output HF model (MI-CHF) and as a high output HF model (AV-CHF), respectively. Blood flow supply to the CB region was decreased only in MI-CHF rats compared to Sham and AV-CHF rats. MI-CHF rats exhibited a significantly enhanced hypoxic ventilatory response compared to AV-CHF rats. However, apnea/hypopnea incidence was similarly increased in both MI-CHF and AV-CHF rats compared to control. Kruppel-like factor 2 expression, a flow sensitive transcription factor, was reduced in the CBs of MI-CHF rats but not in AV-CHF rats. Our results indicate that in the setting of HF, potentiation of the CB chemoreflex is strongly associated with a reduction in cardiac output and may not be related to other pathophysiological consequences of HF.
Assuntos
Corpo Carotídeo/fisiologia , Células Quimiorreceptoras/fisiologia , Insuficiência Cardíaca/fisiopatologia , Reflexo/fisiologia , Animais , Apneia/metabolismo , Apneia/fisiopatologia , Débito Cardíaco/fisiologia , Corpo Carotídeo/metabolismo , Células Quimiorreceptoras/metabolismo , Insuficiência Cardíaca/metabolismo , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/fisiologiaRESUMO
Kruppel-like factors (KLFs) are a group of transcriptional regulators that have recently been identified to exhibit tumor-suppressive function against various gastrointestinal cancers. The present study aims to investigate the expression patterns and prognostic value of KLF-4 in colorectal cancers (CRCs). KLF-4 levels in CRC tissues were examined via immunohistochemistry analysis, real-time quantitative polymerase chain reaction, and western blotting. The chi-square test was performed to evaluate the correlation between KLF-4 expression and the clinicopathological characteristics. Kaplan-Meier analysis was performed to assess the prognostic value of KLF-4 in CRC patients. In addition, we evaluated the effect of KLF-4 knockdown on the proliferation of CRC HT-29 cells. Our results showed significant downregulation of KLF-4 in 31 CRC samples, collected from CRC patients showing more malignant characteristics such as lymphatic metastasis, low tumor cell differentiation, and tumor recurrence. CRC patients in the low KLF-4 group were found to have reduced overall survival and decreased disease-free survival time. Moreover, HT-29 cells transfected with siRNA-KLF-4 showed increased proliferation compared to those transfected with control siRNA. In summary, lower KLF-4 expression was correlated with malignant CRC status and poor prognosis in CRC patients. Moreover, KLF-4 suppression promoted the proliferation of CRC cells in vitro. These results provide novel insights into the tumor suppressive role of KLF-4 in CRC.
Assuntos
Neoplasias Colorretais/patologia , Regulação para Baixo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Diferenciação Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Fator 4 Semelhante a Kruppel , Metástase Linfática , Masculino , Prognóstico , Análise de SobrevidaRESUMO
The Sarco/Endoplasmic Reticulum Ca2+ -ATPases (SERCAs), pump Ca2+ into the endoplasmic reticulum lumen modulating cytosolic Ca2+ concentrations to regulate various cellular processes including cell growth. Previous studies have reported a downregulation of SERCA3 protein expression in gastric and colon cancer cell lines and showed that in vitro cell differentiation increases its expression. However, little is known about the transcriptional mechanisms and transcription factors that regulate SERCA3 expression in epithelial cancer cells. In this work, we demonstrate that SERCA3 mRNA is upregulated up to 45-fold in two epithelial cancer cell lines, KATO-III and Caco-2, induced to differentiate with histone deacetylase inhibitors (HDACi) and by cell confluence, respectively. To evaluate the transcriptional elements responding to the differentiation stimuli, we cloned the human ATP2A3 promoter, generated deletion constructs and transfected them into KATO-III cells. Basal and differentiation responsive DNA elements were located by functional analysis within the first -135 bp of the promoter region. Using site-directed mutagenesis and DNA-protein binding assays we found that Sp1, Sp3, and Klf-4 transcription factors bind to ATP2A3 proximal promoter elements and regulate basal gene expression. We showed that these factors participated in the increase of ATP2A3 expression during cancer cell differentiation. This study provides evidence for the first time that Sp1, Sp3, and Klf-4 transcriptionally modulate the expression of SERCA3 during induction of epithelial cancer cell differentiation. © 2016 Wiley Periodicals, Inc.