RESUMO
ABSTRACT Objective: MCM3AP-AS1 has been characterized as an oncogenic long non-coding RNA (lncRNA) in several cancers including papillary thyroid cancer (PTC), but its role in PTC has not been fully elucidated. Considering the critical role of lncRNAs in cancer biology, further functional analysis of MCM3AP-AS1 in PTC may provide novel insights into PTC management. Subjects and methods: Paired tumor and non-tumor tissues were collected from 63 papillary thyroid carcinoma (PTC) patients. Expression levels of MCM3AP-AS1 , miR-218 and GLUT1 in tissue samples were analyzed by qRT-PCR. Cell transfection was performed to explore the interactions among MCM3AP-AS1 , miR-218 and GLUT1 . Cell proliferation assay was performed to evaluate the effects of MCM3AP-AS1 and miR-218 on cell proliferation. Results: MCM3AP-AS1 accumulated to high levels in PTC tissues and was affected by clinical stage. MCM3AP-AS1 showed a positive correlation with GLUT1 across PTC tissues. RNA interaction prediction showed that MCM3AP-AS1 could bind to miR-218 , which can directly target GLUT1 . MCM3AP-AS1 and miR-218 showed no regulatory role regulating the expression of each other, but overexpression of MCM3AP-AS1 upregulated GLUT1 and enhanced cell proliferation. In contrast, overexpression of miR-218 downregulated GLUT1 and attenuated cell proliferation. In addition, miR-218 suppressed the role of MCM3AP-AS1 in regulating the expression of GLUT1 and cell proliferation. Conclusions: MCM3AP-AS1 may serve as a competing endogenous RNA of miR-218 to upregulate GLUT1 in PTC, thereby promoting cell proliferation. The MCM3AP-AS1/miR-218/GLUT1 pathway characterized in the present study might serve as a potential target to treat PTC.
RESUMO
Objective: MCM3AP-AS1 has been characterized as an oncogenic long non-coding RNA (lncRNA) in several cancers including papillary thyroid cancer (PTC), but its role in PTC has not been fully elucidated. Considering the critical role of lncRNAs in cancer biology, further functional analysis of MCM3AP-AS1 in PTC may provide novel insights into PTC management. Subjects and methods: Paired tumor and non-tumor tissues were collected from 63 papillary thyroid carcinoma (PTC) patients. Expression levels of MCM3AP-AS1, miR-218 and GLUT1 in tissue samples were analyzed by qRT-PCR. Cell transfection was performed to explore the interactions among MCM3AP-AS1, miR-218 and GLUT1. Cell proliferation assay was performed to evaluate the effects of MCM3AP-AS1 and miR-218 on cell proliferation. Results: MCM3AP-AS1 accumulated to high levels in PTC tissues and was affected by clinical stage. MCM3AP-AS1 showed a positive correlation with GLUT1 across PTC tissues. RNA interaction prediction showed that MCM3AP-AS1 could bind to miR-218, which can directly target GLUT1. MCM3AP-AS1 and miR-218 showed no regulatory role regulating the expression of each other, but overexpression of MCM3AP-AS1 upregulated GLUT1 and enhanced cell proliferation. In contrast, overexpression of miR-218 downregulated GLUT1 and attenuated cell proliferation. In addition, miR-218 suppressed the role of MCM3AP-AS1 in regulating the expression of GLUT1 and cell proliferation. Conclusion: MCM3AP-AS1 may serve as a competing endogenous RNA of miR-218 to upregulate GLUT1 in PTC, thereby promoting cell proliferation. The MCM3APAS1/ miR-218/GLUT1 pathway characterized in the present study might serve as a potential target to treat PTC.
Assuntos
Transportador de Glucose Tipo 1 , MicroRNAs , RNA Longo não Codificante , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Transportador de Glucose Tipo 1/genética , Peptídeos e Proteínas de Sinalização Intracelular , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.
Assuntos
Proteína BRCA1 , Dano ao DNA , Proteínas de Ligação a DNA , Enzimas Desubiquitinantes , Chaperonas de Histonas , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Cromatografia Líquida , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HeLa , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Camundongos , Espectrometria de Massas em Tandem , Ubiquitina/metabolismoRESUMO
BACKGROUND: The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. OBJECTIVES: The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. METHODS: We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. RESULTS: Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. CONCLUSIONS: These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.
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Candida glabrata , Proteínas Fúngicas , Antifúngicos/farmacologia , Biofilmes , Candida albicans/metabolismo , Candida glabrata/genética , Candida glabrata/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , VirulênciaRESUMO
PURPOSE: To investigate whether oxymatrine (OMT) prevents hepatic fibrosis in rats by regulating liver transforming growth factor ß1 (TGF-ß1) level. METHODS: Hepatic fibrosis was induced in rats by thioacetamide (TAA). Blood was collected at the end of week 12 to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH). Changes in liver tissue were observed after hematoxylin-eosin (HE) staining. RESULTS: Fibrosis was confirmed by Masson's collagen staining. Liver TGF-ß1 level was determined by ELISA. OMT significantly reduced serum ALT and AST but increased GSH levels in rats with hepatic fibrosis. Moreover, it significantly improved liver histology in rats with TAA-induced hepatic fibrosis. It significantly decreased liver TGF-ß1 level compared to that in the untreated group. It also significantly reduced collagen deposition in rats. CONCLUSION: Oxymatrine is effective in protecting rats from thioacetamide-induced hepatic fibrosis by regulating TGF-ß1 expression.
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Alcaloides/farmacologia , Cirrose Hepática Experimental/prevenção & controle , Substâncias Protetoras/farmacologia , Quinolizinas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Aspartato Aminotransferases/sangue , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/efeitos dos fármacosRESUMO
Abstract Purpose: To investigate whether oxymatrine (OMT) prevents hepatic fibrosis in rats by regulating liver transforming growth factor β1 (TGF-β1) level. Methods: Hepatic fibrosis was induced in rats by thioacetamide (TAA). Blood was collected at the end of week 12 to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH). Changes in liver tissue were observed after hematoxylin-eosin (HE) staining. Results: Fibrosis was confirmed by Masson's collagen staining. Liver TGF-β1 level was determined by ELISA. OMT significantly reduced serum ALT and AST but increased GSH levels in rats with hepatic fibrosis. Moreover, it significantly improved liver histology in rats with TAA-induced hepatic fibrosis. It significantly decreased liver TGF-β1 level compared to that in the untreated group. It also significantly reduced collagen deposition in rats. Conclusion: Oxymatrine is effective in protecting rats from thioacetamide-induced hepatic fibrosis by regulating TGF-β1 expression.
Assuntos
Animais , Masculino , Ratos , Quinolizinas/farmacologia , Substâncias Protetoras/farmacologia , Alcaloides/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Cirrose Hepática Experimental/prevenção & controle , Aspartato Aminotransferases/sangue , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/efeitos dos fármacos , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/metabolismoRESUMO
Purpose: To investigate whether oxymatrine (OMT) prevents hepatic fibrosis in rats by regulating liver transforming growth factor β1 (TGF-β1) level. Methods: Hepatic fibrosis was induced in rats by thioacetamide (TAA). Blood was collected at the end of week 12 to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH). Changes in liver tissue were observed after hematoxylin-eosin (HE) staining. Results: Fibrosis was confirmed by Massons collagen staining. Liver TGF-β1 level was determined by ELISA. OMT significantly reduced serum ALT and AST but increased GSH levels in rats with hepatic fibrosis. Moreover, it significantly improved liver histology in rats with TAA-induced hepatic fibrosis. It significantly decreased liver TGF-β1 level compared to that in the untreated group. It also significantly reduced collagen deposition in rats. Conclusion: Oxymatrine is effective in protecting rats from thioacetamide-induced hepatic fibrosis by regulating TGF-β1 expression.(AU)
Assuntos
Animais , Ratos , Cirrose Hepática , Prevenção de Doenças , Compostos Químicos , Fator de Crescimento Transformador beta1RESUMO
Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.