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1.
Acta cir. bras ; 34(6): e201900602, 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1019265

RESUMO

Abstract Purpose To investigate the role and related mechanisms of miR-106a in sepsis-induced AKI. Methods Serum from sepsis and healthy patients was collected, sepsis mouse model was established by cecal ligation and puncture (CLP). TCMK-1 cells were treated with lipopolysaccharide (LPS) and transfected with THBS2-small interfering RNA (siTHBS2), miR-106a inhibitor, miR-106a mimics and their negative controls (NCs). The expression of miR-106a, thrombospondin 2 (THBS2), Bax, cleaved caspase-3 and Bcl-2, cell viability, relative caspase-3 activity and TNF-α, IL-1β, IL-6 content were respectively detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, Cell Counting Kit-8 (CCK-8) and enzyme linked immunosorbent assay (ELISA). The relationship between miR-106a and THBS2 was confirmed by dual luciferase reporter assay. Results MiR-106a was up-regulated in serum of sepsis patients, CLP-induced mice models and LPS-induced TCMK-1 cells. LPS reduced cell viability and Bcl-2 expression, and increased caspase-3 activity, Bax expression, the content of TNF-α, IL-1β, IL-6. THBS2 was a target of miR-106a. The decreases of caspase-3 activity, TNF-α, IL-1β, IL-6, Bax expression and the increases of cell viability, Bcl-2 expression caused by miR-106a knockdown were reversed when THBS2 silencing in LPS-stimulated TCMK-1 cells. Conclusion MiR-106a aggravated LPS-induced inflammation and apoptosis of TCMK-1 cells via regulating THBS2 expression.


Assuntos
Humanos , Animais , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Ratos , Sepse/patologia , Trombospondinas/farmacologia , MicroRNAs/metabolismo , Células Epiteliais/patologia , Lesão Renal Aguda/metabolismo , Rim/citologia , Ensaio de Imunoadsorção Enzimática , Transfecção , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Apoptose , Sepse/metabolismo , Modelos Animais de Doenças , Lesão Renal Aguda/patologia , Reação em Cadeia da Polimerase em Tempo Real
2.
Electron. j. biotechnol ; 31: 17-23, Jan. 2018. tab, ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1022036

RESUMO

Background: CDC25 is a dual-specificity phosphatase that was first identified in the yeast Schizosaccharomyces pombe as a cell cycle-defective mutant. Although CDC25 is involved in the cell cycle of ovarian granulosa cells, the CDC25 signaling pathway has not been clarified fully. To explore the role of CDC25C in the cell cycle of goat ovarian granulosa cells, a CDC25C-overexpressing vector, pCMV-HA-CDC25C, was constructed and transfected into granulosa cells from adult and young white goats from Jiangsu Nantong. RT-PCR was used to measure CDC25C, CDK1, and WEE1 gene expression levels, and flow cytometry was used to distinguish ovarian granulosa cells in different phases of the cell cycle. Progesterone and estradiol levels in transfected ovarian granulosa cells were also measured. Results: In adult goat follicular granulosa cells transfected with pCMV-HA-CDC25C, CDC25C expression increased significantly, which greatly increased the relative gene expression levels of both CDK1 and WEE1. Additionally, progesterone and estradiol levels were increased in goat follicular granulosa cells overexpressing CDC25C. And the cell cycle results showed that transfection of pCMV-HA-CDC25C leads to a higher proportion of cells in S phase compared to the no vector-transfected groups. Conclusions: The results of this study indicated that the overexpression of CDC25C may increase the gene expression levels of both WEE1 and CDK1 in S phase and accelerate the transition of cells from G1 phase to S phase.


Assuntos
Animais , Feminino , Cabras , Ciclo Celular/fisiologia , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo , Células da Granulosa/enzimologia , Progesterona/análise , Proteínas Tirosina Quinases/genética , Transfecção , Ciclo Celular/genética , Reação em Cadeia da Polimerase/métodos , Apoptose , Quinases Ciclina-Dependentes/genética , Estradiol/análise , Fertilização , Citometria de Fluxo , Fluorescência , Células da Granulosa/metabolismo
3.
Braz. J. Pharm. Sci. (Online) ; 54(1): e00265, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-951915

RESUMO

ABSTRACT In recent years, non-viral delivery systems for plasmid DNA have become particularly important. They can overcome the disadvantages of viral systems such as insertional mutagenesis and unpredicted immunogenicity. Some additional advantages of non-viral gene delivery systems are; good stability, low cost, targetability, delivery of a high amount of genetic materials. The aim of the study was to develop novel non-viral nanosystems suitable for gene delivery. Two formulations were developed for this purpose: water-in-oil microemulsion (ME) and solid lipid nanoparticles (SLN). The microemulsion was composed of Peceol, Tween 80, Plurol oleique, ethanol and water. The SLN was consisting of Precirol, Esterquat-1 (EQ1), Tween 80, Lecithin, ethanol and water. Characterization studies were carried out by measuring particle size, zeta potential, viscosity and pH. TEM imaging was performed on SLN formulations. Protection against DNase I degradation was examined. Cytotoxicity and transfection efficacy of selected formulations were tested on L929 mouse fibroblast cells. Particle sizes of complexes were below 100 nm and with high positive zeta potential. TEM images revealed that SLNs are spherical. The SLN:DNA complexes have low toxicity and good transfection ability. All results showed that the developed SLN formulations can be considered as suitable non-viral gene delivery systems.


Assuntos
DNA/análise , Genes/genética , Transfecção/estatística & dados numéricos , Terapia Genética/classificação
4.
Braz. j. med. biol. res ; 51(6): e7080, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889093

RESUMO

Prostate cancer (PCa) is the second leading cause of cancer death in men. Irradiation is one of the available options for treatment of PCa, however, approximately 10-45% of PCa are resistant to irradiation. We aimed to explore the role of long non-coding RNA highly upregulated in liver cancer (HULC) in the sensitivity of PCa cells to irradiation. Survival rate, cell apoptosis, cycle, expressions of related proteins, and caspase-3 activity were assessed to explore the effects of HULC on sensitivity of PCa cells to irradiation. Expression of HULC in DU-145, PC3, LNCaP, and RWPE-1 cells was determined and the influence of HULC on DU-145 cells was explored. Then, PC3 cells aberrantly expressing HULC were implanted into NOD-SCID mice for tumor xenograft study. Changes of autophagy after aberrant expression of HULC in vivo and in vitro were tested. Furthermore, the interacted protein of HULC and involved signaling pathway were investigated. In PC3 and LNCaP cells under irradiation, survival rate and cell cycle were decreased and apoptosis was increased by HULC knockdown. HULC knockdown arrested PC3 cells at G0/G1 phase. DU-145 was sensitive to irradiation, and resistance to irradiation of DU-145 cells was enhanced by HULC overexpression. Moreover, HULC knockdown enhanced the sensitivity of PC3 xenografts to irradiation. HULC knockdown promoted autophagy through interaction with Beclin-1 and inhibition of mTOR, resulting in increased apoptosis. HULC knockdown improved sensitivity of PCa cells to irradiation both in vivo and in vitro. HULC suppressed Beclin-1 phosphorylation, thereby reduced autophagy, involving the mTOR pathway.


Assuntos
Humanos , Masculino , Autofagia/efeitos da radiação , Neoplasias da Próstata/patologia , Tolerância a Radiação/fisiologia , RNA Longo não Codificante/efeitos da radiação , Apoptose/efeitos da radiação , Western Blotting , Linhagem Celular Tumoral/efeitos da radiação , Reação em Cadeia da Polimerase em Tempo Real , Interferência de RNA/efeitos da radiação , Transfecção
5.
Braz. j. med. biol. res ; 51(6): e7046, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889094

RESUMO

Thyroid cancer is a common malignant tumor. Long non-coding RNA colon cancer-associated transcript 1 (lncRNA CCAT1) is highly expressed in many cancers; however, the molecular mechanism of CCAT1 in thyroid cancer remains unclear. Hence, this study aimed to investigate the effect of CCAT1 on human thyroid cancer cell line FTC-133. FTC-133 cells were transfected with CCAT1 expressing vector, CCAT1 shRNA, miR-143 mimic, and miR-143 inhibitor, respectively. After different treatments, cell viability, proliferation, migration, invasion, and apoptosis were measured. Moreover, the regulatory relationship of CCAT1 and miR-143, as well as miR-143 and VEGF were tested using dual-luciferase reporter assay. The relative expressions of CCAT1, miR-143, and VEGF were tested by qRT-PCR. The expressions of apoptosis-related factors and corresponding proteins in PI3K/AKT and MAPK pathways were analyzed using western blot analysis. The results suggested that CCAT1 was up-regulated in the FTC-133 cells. CCAT1 suppression decreased FTC-133 cell viability, proliferation, migration, invasion, and miR-143 expression, while it increased apoptosis and VEGF expression. CCAT1 might act as a competing endogenous RNA (ceRNA) for miR-143. Moreover, CCAT1 activated PI3K/AKT and MAPK signaling pathways through inhibition of miR-143. This study demonstrated that CCAT1 exhibited pro-proliferative and pro-metastasis functions on FTC-133 cells and activated PI3K/AKT and MAPK signaling pathways via down-regulation of miR-143. These findings will provide a possible target for clinical treatment of thyroid cancer.


Assuntos
Humanos , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias da Glândula Tireoide/patologia , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Invasividade Neoplásica/patologia , RNA Longo não Codificante/genética , Transfecção
6.
Braz. j. med. biol. res ; 51(6): e6452, 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889104

RESUMO

Several microRNAs (miRNAs) have been reported as oncogenes or tumor suppressors in many cancers, including gastric cancer (GC). However, the role and molecular mechanism of miR-3129 in GC is largely unknown. We aimed to explore the function and the underlying molecular mechanism of miR-3129 in GC. Cancer tissues and corresponding adjacent tissues were collected from 50 patients with GC, and the expression of miR-3129 was detected by RT-qPCR. The expression of miR-3129 and pRb in human GC cell line SCG7091 was altered by transient transfection. Thereafter, MTT and flow cytometry assays were used to analyze cell viability and cell cycle. The expression of cyclin E, CDK2, CDK2 inhibitors (p16 and 21), and pRb were detected by RT-qPCR and western blot. A significant up-regulation of miR-3129 was observed in GC tissues compared to adjacent tissues. Overexpression of miR-3129 significantly improved cell viability after 4 days of post-transfection. Flow cytometry assay results showed that the miR-3129 overexpression arrested more SGC7901 cells at S phase. Moreover, overexpression of miR-3129 down-regulated the expression of CDK2 inhibitors while it up-regulated the expression levels of cyclin E, CDK2, and pRb. Interestingly, we found that pRb inhibition reversed the effect of miR-3129 inhibitor on cell proliferation in SGC7901 cells, increased cell viability, reduced cells at G0/1 phase, and modulated the expression of proliferation-related factors. Our results revealed that miR-3129 functioned as an oncogene through positive regulation of pRb and may prove to be a promising option for molecular therapy of GC.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Proliferação de Células/genética , Proteína do Retinoblastoma/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação para Baixo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase em Tempo Real , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transfecção , Regulação para Cima
7.
Braz. j. med. biol. res ; 51(6): e7061, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889105

RESUMO

Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.


Assuntos
Animais , Camundongos , Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Diterpenos/farmacologia , Subunidade beta da Proteína Ligante de Cálcio S100/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Astrócitos/fisiologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Fatores de Tempo , Transfecção
8.
Braz. j. med. biol. res ; 51(6): e6555, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889109

RESUMO

Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cardiovascular diseases, especially in myocardial infarction and ischemia/reperfusion (I/R). However, the underlying molecular mechanism remains unclear. In this study, we determined the role and the possible underlying molecular mechanism of lncRNA-ROR in myocardial I/R injury. H9c2 cells and human cardiomyocytes (HCM) were subjected to either hypoxia/reoxygenation (H/R), I/R or normal conditions (normoxia). The expression levels of lncRNA-ROR were detected in serum of myocardial I/R injury patients, H9c2 cells, and HCM by qRT-PCR. Then, levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured by kits. Cell viability, apoptosis, apoptosis-associated factors, and p38/MAPK pathway were examined by MTT, flow cytometry, and western blot assays. Furthermore, reactive oxygen species (ROS) production was determined by H2DCF-DA and MitoSOX Red probes with flow cytometry. NADPH oxidase activity and NOX2 protein levels were measured by lucigenin chemiluminescence and western blot. Results showed that lncRNA-ROR expression was increased in I/R patients and in H/R treatment of H9c2 cells and HCM. Moreover, lncRNA-ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX. Furthermore, lncRNA-ROR decreased cell viability, increased apoptosis, and regulated expression of apoptosis-associated factors. Additionally, lncRNA-ROR increased phosphorylation of p38 and ERK1/2 expression and inhibition of p38/MAPK, and rescued lncRNA-ROR-induced cell injury in H9c2 cells and HCM. ROS production, NADPH oxidase activity, and NOX2 protein levels were promoted by lncRNA-ROR. These data suggested that lncRNA-ROR acted as a therapeutic agent for the treatment of myocardial I/R injury.


Assuntos
Humanos , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , RNA Longo não Codificante/metabolismo , Apoptose , Western Blotting , Sobrevivência Celular , Glutationa Peroxidase/metabolismo , Hidroliases/metabolismo , Malondialdeído/metabolismo , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos , Reação em Cadeia da Polimerase em Tempo Real , RNA Longo não Codificante/genética , Transdução de Sinais , Superóxido Dismutase/metabolismo , Transfecção
9.
Braz. j. med. biol. res ; 51(6): e6997, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889113

RESUMO

MicroRNAs (miRNAs) have been reported to be associated with heart valve disease, which can be caused by inflammation. This study aimed to investigate the functional impacts of miR-27a on TNF-α-induced inflammatory injury in human mitral valve interstitial cells (hMVICs). hMVICs were subjected to 40 ng/mL TNF-α for 48 h, before which the expressions of miR-27a and NELL-1 in hMVICs were altered by stable transfection. Trypan blue staining, BrdU incorporation assay, flow cytometry detection, ELISA, and western blot assay were performed to detect cell proliferation, apoptosis, and the release of proinflammatory cytokines. We found that miR-27a was lowly expressed in response to TNF-α exposure in hMVICs. Overexpression of miR-27a rescued hMVICs from TNF-α-induced inflammatory injury, as cell viability and BrdU incorporation were increased, apoptotic cell rate was decreased, Bcl-2 was up-regulated, Bax and cleaved caspase-3/9 were down-regulated, and the release of IL-1β, IL-6, and MMP-9 were reduced. NELL-1 was positively regulated by miR-27a, and NELL-1 up-regulation exhibited protective functions during TNF-α-induced cell damage. Furthermore, miR-27a blocked JNK and Wnt/β-catenin signaling pathways, and the blockage was abolished when NELL-1 was silenced. This study demonstrated that miR-27a overexpression protected hMVICs from TNF-α-induced cell damage, which might be via up-regulation of NELL-1 and thus modulation of JNK and Wnt/β-catenin signaling pathways.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Inflamação/induzido quimicamente , MicroRNAs/metabolismo , Valva Mitral/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Proliferação de Células , Sobrevivência Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Doenças das Valvas Cardíacas/prevenção & controle , Inflamação/patologia , Valva Mitral/citologia , Valva Mitral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Regulação para Cima
10.
Braz. j. med. biol. res ; 51(5): e7319, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-889079

RESUMO

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.


Assuntos
Animais , Masculino , Ratos , Traumatismo por Reperfusão/metabolismo , Isquemia do Cordão Espinal/metabolismo , MicroRNAs/metabolismo , Sirtuína 1/metabolismo , Transfecção , Traumatismo por Reperfusão/fisiopatologia , Regulação para Baixo/fisiologia , Regulação para Cima/fisiologia , Western Blotting , Ratos Sprague-Dawley , Apoptose , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Isquemia do Cordão Espinal/fisiopatologia , Modelos Animais de Doenças , Citometria de Fluxo
11.
Clin. biomed. res ; 37(4): 330-333, 2017. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-876699

RESUMO

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)


Assuntos
Humanos , Fibroblastos/enzimologia , Técnicas de Transferência de Genes , Vetores Genéticos , Iduronidase/metabolismo , Mucopolissacaridose I/terapia , DNA Complementar , Terapia Genética/métodos , Iduronidase/genética , Mucopolissacaridose I/genética , Plasmídeos/genética , Transfecção/métodos
12.
Braz. J. Pharm. Sci. (Online) ; 53(3): e00140, 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889394

RESUMO

ABSTRACT Cationic polymers such as polyallylamine (PAA) having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P) ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa) were suitable vectors for gene delivery with low cytotoxicity.


Assuntos
Terapia Genética , Colesterol/uso terapêutico , Polímeros , Transfecção/instrumentação
13.
Braz. j. med. biol. res ; 50(4): e5861, 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-839274

RESUMO

Myocardial ischemia is a major cause of death and remains a disease with extremely deficient clinical therapies and a major problem worldwide. Cold inducible RNA-binding protein (CIRBP) is reported to be involved in multiple pathological processes, including myocardial ischemia. However, the molecular mechanisms of myocardial ischemia remain elusive. Here, we first overexpressed CIRBP by transfection of pc-CIRBP (pcDNA3.1 containing coding sequenced for CIRBP) and silenced CIRBP by transfection of small interfering RNA targeting CIRBP (siCIRBP). pcDNA3.1 and the negative control of siCIRBP (siNC) were transfected into H9C2 cells to act as controls. We then constructed a cell model of myocardial ischemia through culturing cells in serum-free medium with hypoxia in H9C2 cells. Subsequently, AlamarBlue assay, flow cytometry and western blot analysis were used, respectively, to assess cell viability, reactive oxygen species (ROS) level and apoptosis, and expression levels of IκBα, p65 and Bcl-3. We demonstrated that CIRBP overexpression promoted cell proliferation (P<0.001), inhibited cell apoptosis (P<0.05), reduced ROS level (P<0.001), down-regulated phosphorylated levels of IκBα and p65 (P<0.01 or P<0.001), and up-regulated expression of Bcl-3 (P<0.001) in H9C2 cells with myocardial ischemia. The influence of CIRBP knockdown yielded opposite results. Our study revealed that CIRBP could protect H9C2 cells against myocardial ischemia through inhibition of NF-κB pathway.


Assuntos
Animais , Ratos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , NF-kappa B/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Proteínas de Ligação a RNA/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/análise , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Tempo , Transfecção/métodos
14.
Braz. j. med. biol. res ; 50(6): e6103, 2017. graf
Artigo em Inglês | LILACS | ID: biblio-839312

RESUMO

Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3′UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.


Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Neoplasias Colorretais/enzimologia , Histona Desacetilase 2/metabolismo , MicroRNAs/metabolismo , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Neoplasias Colorretais/genética , Regulação para Baixo , Células HCT116 , Histona Desacetilase 2/genética , MicroRNAs/genética , Transfecção , Regulação para Cima
15.
Biomédica (Bogotá) ; 36(supl.1): 128-136, dic. 2016. ilus, graf
Artigo em Espanhol | LILACS | ID: lil-783530

RESUMO

Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.


Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.


Assuntos
Giardia lamblia , Spliceossomos , Parasitos , Processamento de RNA , Transfecção , Organismos Eucariotos Unicelulares
16.
Mem. Inst. Oswaldo Cruz ; 111(7): 460-468, tab, graf
Artigo em Inglês | LILACS | ID: lil-787557

RESUMO

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.


Assuntos
Animais , Feminino , Proteínas de Choque Térmico HSP70/fisiologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/fisiologia , Estresse Fisiológico , Proteínas de Choque Térmico HSP70/genética , Leishmania mexicana/genética , Leishmania mexicana/ultraestrutura , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/fisiologia , Estresse Oxidativo , Proteínas de Protozoários/genética , Transfecção/métodos
17.
São Paulo; s.n; 2016. [147] p. ilus, tab, graf.
Tese em Português | LILACS | ID: biblio-870899

RESUMO

O câncer de próstata (CaP) é o tumor mais comum do homem nos países ocidentais e a segunda causa de óbito por câncer em homens nos EUA, Europa e Brasil. O câncer localizado tem sobrevida câncer especifica elevada quando tratado adequadamente, porém a doença metastática ainda apresenta tratamentos pouco eficientes com sobrevida global de 28%. Os microRNAs (miRNAs) são um grupo de moléculas pequenas de RNA que contém entre 19 a 25 nucleotídeos não codificantes de proteína, com ação fundamental na regulação da expressão gênica. Eles estão envolvidos em processos essenciais nas células normais e neoplásicas como ciclo celular, proliferação, apoptose, metabolismo energético, invasão e metastatização. Objetivos: Realizar estudos in vitro e in vivo usando miRNA em um modelo de câncer de próstata metastático inédito no nosso meio com intuito de analisar o seu potencial como agente terapêutico dessa neoplasia. Métodos: Nos estudos in vitro, três linhagens celulares foram utilizadas (PC3, DU145 e LNCaP). Essas linhagens foram transfectadas com os miRNAs 100, 145 e 373 e seus respectivos antiMiRs utilizando-se lipofectamina. Analisamos a expressão dos genes alvo mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 por PCR quantitativo em tempo real (qRT-PCR). Foram realizados também estudos de apoptose, ciclo celular e ploidia utilizando o citômetro de fluxo. Alterações no potencial de invasão foram avaliadas pela técnica do matrigel. O modelo in vivo pré-clínico foi desenvolvido pela injeção intra-cardíaca da linhagem PC-3M-Luc-C6 em camundongos NUDE com 9 semanas. O crescimento tumoral foi avaliado com o sistema de bioluminescência in vivo. Após o pleno estabelecimento das metástases no dia 21, os animais foram tratados com três injeções na veia da cauda contendo o miRNA conjugado com o atelocolágeno. Os animais foram sacrificados e no dia 48 para análise dos tecidos. Resultados: miR-100 aumenta a apoptose na LNCaP, e reduz a apoptose na DU145. Na linhagem DU145...


Prostate cancer (PCa) is the most common neoplasia of man in Western countries and the second cause of death by cancer in men in the US, Europe and Brazil. The localized cancer has high cancer-specific survival when treated properly, however metastatic disease still presents low effective treatments with 28% of global survival. microRNAs (miRNAs) are a group of small RNA molecules containing from 19 to 25 nucleotides of noncoding protein with fundamental action in the regulation of gene expression. They are involved in key processes in normal and neoplastic cells as cell cycle, proliferation, apoptosis, energy metabolism, invasion and metastasis. Objectives: To carry out studies in vitro and in vivo using miRNA in a novel model of metastatic prostate cancer in our country in order to evaluate its potential as a therapeutic agent of this neoplasia. Methods: In the in vitro studies, three cell lines were used (PC3, DU145 and LNCaP). These cell lines were transfected with miRNAs 100, 145 and 373 and their antiMiRs using lipofectamine. We analyzed the gene expression of mTOR, SMARCA5, KRAS, CMYC, MMP9, CD44 by real-time polymerase chain reaction (qRT-PCR). We also performed studies of apoptosis, cell cycle and ploidy using flow cytometer. Changes in the invasion potential were evaluated by the technique of matrigel. The pre-clinical model in vivo was developed by intracardiac injection of PC-3MLuc-C6 cell line in NUDE mice with 9 weeks. Tumor growth was evaluated with an in vivo image system (IVIS). After the full establishment of metastases on day 21, the animals were treated with three injections into the tail vein containing the miRNA plus atelocollagen. The animals were sacrificed on day 48 for tissues analysis. Results: MiR-100 increases apoptosis in LNCaP and reduces apoptosis in DU145. The anti-miR-100 increased apoptosis in 14% in PC3. In cell line DU145, miR-100 inhibited proliferation. In the analysis of gene expression, the miR-100...


Assuntos
Animais , Masculino , Camundongos , Apoptose , Ciclo Celular , Expressão Gênica , MicroRNAs , Modelos Animais , Imagem Molecular , Metástase Neoplásica , Neoplasias da Próstata , Terapêutica , Transfecção , Biologia Molecular
18.
Braz. j. med. biol. res ; 48(12): 1063-1070, Dec. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-762916

RESUMO

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.


Assuntos
Humanos , Condrócitos/metabolismo , Colágeno Tipo II/biossíntese , Glicosaminoglicanos/biossíntese , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Matrilinas/biossíntese , Fatores de Transcrição SOX9/metabolismo , Transfecção/métodos , Cartilagem Articular/lesões , Cartilagem Articular/metabolismo , Colágeno Tipo II/análise , Matriz Extracelular/química , Expressão Gênica , Glicosaminoglicanos/análise , Fator de Crescimento Insulin-Like I/genética , Proteínas Matrilinas/genética , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/metabolismo , Fatores de Transcrição SOX9/genética , Espectrofotometria
19.
Rev. chil. pediatr ; 86(4): 251-256, ago. 2015. tab
Artigo em Espanhol | LILACS | ID: lil-764081

RESUMO

Introducción: Autoconcepto es el conjunto de ideas y actitudes que se tiene acerca de sí mismo. Nuestro objetivo fue evaluar si existen diferencias en el nivel de autoconcepto de niños de 8-12 años con y sin secuelas de quemaduras, e identificar variables predictoras del autoconcepto en los niños con secuelas. Pacientes y método: Estudio comparativo, transversal de 109 niños con secuelas de quemaduras de 8 a 12 años de edad, con 109 niños sin secuelas de quemaduras, del mismo grupo de edad y nivel socioeconómico. Se utilizó la escala de autoconcepto de Piers-Harris, que entrega medida de autoconcepto general y dimensiones: conductual, estatus intelectual y escolar, apariencia y atributos físicos, ansiedad, popularidad, felicidad y satisfacción. Resultados: No hubo diferencias significativas en el nivel de autoconcepto general ni en sus dimensiones al comparar ambos grupos (p > 0,05). Dentro del grupo con secuelas de quemaduras la variable número de secuelas resultó ser un factor protector para las dimensiones ansiedad, popularidad, felicidad-satisfacción y autoconcepto general. La variable localización surgió como factor de riesgo para la dimensión conductual. Discusión: La ausencia de diferencias en autoconcepto entre niños con secuelas de quemaduras y sin ellas es similar a lo reportado por la literatura. El hallazgo en factores de riesgo y protectores motiva a continuar investigando, incorporando antecedentes premórbidos y familiares.


Introduction: Self-concept is the set of ideas and attitudes that a person has about him/herself. Objective: To evaluate whether there are differences in the level of self-concept in children 8-12 years old with and without burns sequelae. To identify predictive variables of self-concept in children with sequelae. Patients and method: A comparative cross-sectional study of self-concept in 109 children with burns sequelae, from 8 to 12 years old, with 109 children without burns sequelae, and of the same age and socioeconomic status. The Piers-Harris self-concept scale is used, which provides a general measurement of self-concept and behavioural, intellectual and school status, appearance, and physical attributes, anxiety, popularity, happiness and satisfaction dimensions. Results: There were no significant differences in the level of general self-concept or their dimensions (P > .05). In the group with burns sequelae, the protective factor was the variable number of sequels was associated with the dimensions of anxiety, popularity, happiness-satisfaction and general self-concept. The location variable emerged as a risk factor for the behavioural dimension. Discussion: The absence of differences in self-concept between children with burns sequelae and children without them is similar to that reported in the literature. The finding in the risk and protective factors encourages to further research, and perhaps incorporating pre-morbidity and family background.


Assuntos
Humanos , DNA , Compostos de Amônio Quaternário/química , Serina/química , Tensoativos/química , Amidas/química , Aminas/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Ésteres/química , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HeLa , Lipídeos/química , Transfecção/métodos
20.
Rev. chil. pediatr ; 86(4): 287-290, ago. 2015. ilus, graf
Artigo em Espanhol | LILACS | ID: lil-764087

RESUMO

Introducción: La telorragia es un síntoma poco frecuente en pacientes pediátricos, la causa más frecuente en esta población es la ectasia ductal mamaria (EDM), que es una afección benigna y autolimitada, caracterizada por la dilatación del conducto mamario, fibrosis e inflamación periductal. Objetivo: Presentar un caso de EDM, para facilitar el rápido reconocimiento por parte de los médicos, y evitar estudios y tratamientos agresivos. Caso clínico: Lactante de sexo masculino de 6 meses de edad, sano, alimentado por lactancia materna exclusiva; consultó por un nódulo retroareolar derecho y telorragia unilateral. Se realizó una ecografía Doppler que mostró una lesión multiquística, sugerente de una EDM. Se planteó tratamiento expectante y acudió a control a los 6 meses con excelente evolución. Conclusiones: La EDM es la principal causa de telorragia en niños, corresponde a una afección benigna, y la resolución generalmente es espontánea, antes de los 9 meses. Por lo que su conocimiento es de gran relevancia para el adecuado diagnóstico y manejo de estos pacientes.


Introduction: Bloody nipple discharge is an infrequent symptom during childhood. The most common cause in this population is mammary duct ectasia (MDE), which is a benign and self-limiting condition, that is characterized by dilatation of the mammary ducts, fibrosis and periductal inflammation. Objective: Report of a case of MDE in order to improve physicians’ diagnosis accuracy and avoid aggressive studies and treatments. Case report: Six-months old male healthy infant, exclusively breastfeeded, that visited our clinic with a lump beneath his right nipple and bloody discharge from the same nipple. An ultrasound was performed which showed a multicystic lesion suggestive of MDE. Watchful waiting was decided as treatment, with good evolution after six months of follow up. Conclusions: The MDE is the leading cause of bloody discharge in pediatric population, being a benign condition that resolves spontaneously before nine months. The knowledge of this condition is essential so as to accurately diagnose and treat it.


Assuntos
Humanos , Cátions/química , Indicadores e Reagentes/química , Lipídeos/química , Polienos/química , RNA Interferente Pequeno/química , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HeLa , Lipossomos/química , Luciferases/química , Fosfolipídeos/química , RNA Interferente Pequeno/genética , Transfecção/métodos
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