Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 30
Filter
1.
Int. j. morphol ; 38(6): 1639-1644, Dec. 2020. graf
Article in English | LILACS | ID: biblio-1134491

ABSTRACT

SUMMARY: Previous studies from our group described the consequences of using ethanol on penile erection. Nevertheless, the molecular mechanisms surrounding microRNAs, apoptosis process and their relationship with erectile dysfunction associated with alcohol consumption are still poorly understood. The objective of this analysis was to evaluate the mechanism of apoptosis by the expression of AIF and PARP, as well as their regulatory microRNAs: miR-145, miR-210 and miR-486, in the corpus cavernosum of rats submitted to a semivoluntary alcoholism model. For this study 24 Wistar rats were divided into two groups: control (C) and treated with 20 % ethanol (A) for seven weeks. The corpus cavernosum samples were prepared for immunohistochemical analysis of AIF and PARP protein expression, and microRNAs miR-145, miR-210, miR-486 gene expression in cavernous tissue was performed by real time PCR. The immunohistochemical analysis showed little nuclear positive labeling for the protein PARP and AIF in the corpus cavernosum of control and ethanol treated animals. After analysis of miR-145, -210 and -486 microRNA expression in the 12 animals studied, no results were found with significant statistical difference between the control and alcoholized groups. The expression of AIF and PARP and their regulatory microRNAs involved in apoptotic process (miR-145, miR-210 and miR-486) were not altered in the corpus cavernosum of rats submitted to semivoluntary alcoholism.


RESUMEN: Estudios previos de nuestro grupo describieron las consecuencias del uso de etanol en la erección del pene. Sin embargo, los mecanismos moleculares que rodean a los microARN, el proceso de apoptosis y su relación con la disfunción eréctil asociada con el consumo de alcohol aún no se conocen bien. El objetivo de este análisis fue evaluar el mecanismo de apoptosis mediante la expresión de AIF y PARP, así como sus microARN reguladores: miR-145, miR-210 y miR-486, en el cuerpo cavernoso de ratas sometidas a un modelo de alcoholismo semivoluntario. Se dividieron 24 ratas Wistar en dos grupos: control (C) grupo de ratas tratadas con etanol al 20 % (A) durante siete semanas. Las muestras del cuerpo cavernoso se prepararon para el análisis inmunohistoquímico de la expresión de la proteína AIF y PARP, y la expresión del gen microRNAs miR-145, miR-210, miR-486 en tejido cavernoso se realizó por PCR en tiempo real. El análisis inmunohistoquímico mostró escaso etiquetado nuclear positivo para la proteína PARP y AIF en el cuerpo cavernoso de los animales de control y tratados con etanol. Después del análisis de la expresión de microARN miR-145, -210 y -486 no se encontraron resultados con diferencias estadísticas significativas entre los grupos control y alcoholizados. La expresión de AIF y PARP y sus microARN reguladores involucrados en el proceso apoptótico (miR-145, miR-210 y miR-486) no se alteraron en el cuerpo cavernoso de las ratas sometidas a alcoholismo semivoluntario.


Subject(s)
Animals , Rats , Apoptosis , Alcoholism/metabolism , Erectile Dysfunction/metabolism , Penis/physiopathology , Penis/chemistry , Immunohistochemistry , Rats, Wistar , MicroRNAs/analysis , MicroRNAs/genetics , MicroRNAs/metabolism , Disease Models, Animal , Alcoholism/physiopathology , Apoptosis Inducing Factor/analysis , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Real-Time Polymerase Chain Reaction , Erectile Dysfunction/physiopathology
2.
Braz. oral res. (Online) ; 34: e002, 2020. tab, graf
Article in English | LILACS | ID: biblio-1055528

ABSTRACT

Abstract Oral potentially malignant disorders (OPMD) possess significant chances of malignancy conversion. In order to develop an early diagnostic tool, the present study evaluated the expression of miRNA-21 and 31 as salivary markers. The case-control study was carried out in 36 healthy participants as controls and in 36 patients who were newly diagnosed as OPMD having four different lesions including leucoplakia, oral sub mucous fibrosis (OSMF)궱, oral lichen planus, and (OSMF)궱 with leucoplakia. The samples were also classified as non-dysplastic, or with mild, moderate, and severe dysplasia according to their histopathological reports. The salivary miRNA-21 and 31 expressions were studied using real-time PCR. The statistical analysis was carried out using SPSS version 22. Salivary miRNA-21 (p-value = 0.02) and 31 (p-value = 0.01) were significantly upregulated in severe dysplasia compared with control. Among the different lesions, leucoplakia had significant upregulation of miRNA-21 and 31. miRNA-21 can be used as a diagnostic marker with specificity of 66% and sensitivity of 69%. The area under the ROC curve was 0.820 for miRNA-21 and 0.5 for miRNA-31, which proved that miRNA-21 is a better diagnostic marker than miRNA-31 for OPMD.


Subject(s)
Humans , Precancerous Conditions/pathology , Saliva/chemistry , Mouth Neoplasms/pathology , MicroRNAs/analysis , Oral Submucous Fibrosis/pathology , Reference Values , Severity of Illness Index , Leukoplakia, Oral/pathology , Mouth Neoplasms/chemistry , Biomarkers, Tumor/analysis , Case-Control Studies , Linear Models , ROC Curve , Analysis of Variance , Lichen Planus, Oral/pathology , Real-Time Polymerase Chain Reaction
3.
J. appl. oral sci ; 28: e20190382, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1056584

ABSTRACT

Abstract Objective This study aimed to investigate the effects of Maras powder (a type of smokeless tobacco obtained from Nicotiana rustica Linn and mixed with the ashes of wood, especially from oak, walnut or grapevine) on the microRNA (miRNA) deregulation of oral mucosa, and it compares these effects with those of smoking. Methodology Oral mucosal samples were collected from 74 patients, consisting of 16 nonusers, 26 smokers, and 32 Maras powder users. Genes associated with oral cancer were selected and 90 microRNAs targeting these genes were identified. MicroRNA were isolated and purified using the microRNA isolation kit. MicroRNA were expressed using Fluidigm RT-PCR. Results A positive correlation between the duration of Maras powder use with miR-31 expression levels, and a negative correlation between the Maras powder chewing time and miR-372 expression levels was found. In addition, there is a negative correlation between the amount of Maras powder consumed and expression levels of miR-375, miR-378a, miR-145, and miR-10b; moreover, another negative correlation is observed between the number of cigarettes consumed and the expression levels of miR-23a, miR-23b, miR-203a, miR-200b, and miR-375. However, miR-200b and miR-92a levels were downregulated significantly more in Maras powder users when compared with smokers and nonusers (p<0.05). Conclusion The results show both chewing Maras powder and smoking have an effect on deregulation of miR-200b and miR-92a expressions. This leads to the belief that assessing the expression of these two miRNAs is a promising noninvasive method of analysis, especially in mutagen exposures. Finally, large-scale and high-throughput studies may help to identify an extensive miRNA expression profile associated with tobacco use and improve the understanding of oral malignancies.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Tobacco, Smokeless/adverse effects , MicroRNAs/drug effects , Mouth Mucosa/drug effects , Powders , Time Factors , Mouth Neoplasms/genetics , Down-Regulation , Gene Expression , Cross-Sectional Studies , Risk Factors , Analysis of Variance , MicroRNAs/analysis , Real-Time Polymerase Chain Reaction
4.
J. bras. nefrol ; 41(2): 242-251, Apr.-June 2019. tab, graf
Article in English | LILACS | ID: biblio-1012532

ABSTRACT

ABSTRACT Introduction: The development of novel non-invasive biomarkers of kidney graft dysfunction, especially in the course of the delayed graft function period would be an important step forward in the clinical practice of kidney transplantation. Methods: We evaluated by RT-PCR the expression of miRNA-146 to -5p ribonucleic micro-acids (miRNAs) in the peripheral blood and renal tissue obtained from kidney transplant recipients who underwent a surveillance graft biopsy during the period of delayed graft function. Results: In biopsy samples, the expression of miR-146a-5p was significantly increased in the group of patients with delayed graft function (DGF) (n = 33) versus stables patients (STA) (n = 13) and patients with acute rejection (AR) (n = 9) (p = 0.008). In peripheral blood samples, a non-significant increase of miR-146a-5p expression was found in the DGF group versus STA and AR groups (p = 0.083). No significant correlation was found between levels of expression in biopsy and plasma. ROC curve analysis revealed an AUC of 0.75 (95% CI: 0.62-0.88) for the renal tissue expression and 0.67 (95% CI 0.52-0.81) for the peripheral blood expression. Conclusion: We conclude that miR-146a-5p expression has a distinct pattern in the renal tissue and perhaps in the peripheral blood in the setting of DGF. Further refinements and strategies for studies should be developed in the field of non-invasive molecular diagnosis of kidney graft dysfunction.


RESUMO Introdução: O desenvolvimento de novos biomarcadores não invasivos para disfunção do enxerto renal, especialmente no decurso da disfunção inicial do enxerto, seria de enorme valia para a prática clínica do transplante renal. Métodos: A técnica de RT-PCR foi utilizada para avaliar a expressão de microRNA 146a-5p no sangue periférico e no tecido renal de receptores de transplante submetidos a biópsia renal de controle no decurso de disfunção inicial do enxerto. Resultados: A expressão de miR-146a-5p estava significativamente aumentada nas amostras de biópsia do grupo de pacientes com disfunção inicial do enxerto (DIE) (n = 33) em relação aos pacientes estáveis (n = 13) e aos com rejeição aguda (RA) (n = 9) (p = 0,008). Foi detectado aumento não significativo da expressão de miR-146a-5p nas amostras de sangue periférico do grupo com DIE em comparação aos pacientes estáveis e com RA (p = 0,083). Não foi identificada correlação significativa entre os níveis de expressão no plasma e na biópsia. A análise da curva COR revelou uma ASC de 0,75 (IC 95%: 0,62-0,88) para a expressão no tecido renal e de 0,67 (IC 95% 0,52-0,81) no sangue periférico. Conclusão: A expressão de miR-146a-5p tem um padrão distinto no tecido renal e talvez no sangue periférico em cenários de DIE. Maiores refinamentos e estratégias adicionais de estudo devem ser desenvolvidos na área do diagnóstico molecular não invasivo da disfunção do enxerto renal.


Subject(s)
Humans , Male , Female , Adult , Kidney Transplantation , MicroRNAs/genetics , Delayed Graft Function/genetics , Biopsy , Biomarkers/analysis , Reperfusion Injury/genetics , ROC Curve , Area Under Curve , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/analysis , Delayed Graft Function/pathology , Graft Rejection/genetics
5.
Arq. bras. cardiol ; 112(2): 154-162, Feb. 2019. tab, graf
Article in English | LILACS | ID: biblio-983823

ABSTRACT

Abstract Background: Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment . MiR-126 and miR-210 support angiogenic response in endothelial cells. Objective: The present study sought to explore the effect of garlic and voluntary exercise, alone or together, on miR-126 and miR-210 expressions and cardiac angiogenesis in rats with type 1 diabetes. Methods: Male Wistar rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Diabetes was induced in the animals by streptozotocin (ip, 50 mg/kg). The rats were then fed raw fresh garlic homogenate (250 mg/kg) or were subjected to voluntary exercise, or to combined garlic and voluntary exercise for 6 weeks. MiR-126 and miR-210 expressions in the myocardium were determined by real time PCR, and the serum lipid profile was measured by enzymatic kits. Angiogenesis was evaluated by immunostaining for PECAM-1/ CD31 in the myocardium. Results: Diabetes reduced both cardiac miR-126 expression and angiogenesis (p < 0.05). On the other hand, there was a miR-210 expression increase in the myocardium of diabetic animals (p < 0.001). However, those effects reversed either with garlic or voluntary exercise (p < 0.01). Moreover, treating diabetic rats with garlic and voluntary exercise combined had an additional effect on the expressions of miR-126 and miR-210 (p < 0.001). Furthermore, both voluntary exercise and garlic significantly improved serum lipid profiles (p < 0.001). Conclusion: The induction of diabetes decreased angiogenesis in the myocardium, whereas our treatment using long-term voluntary exercise and garlic improved myocardial angiogenesis. These changes were possibly owing to the enhancement of myocardial miR-126 and miR-210 expressions.


Resumo Fundamento: O diabetes mellitus (DM) é um dos principais fatores de risco para doenças cardiovasculares, levando à disfunção endotelial e inibição da angiogênese. O miRNA-126 e o miRNA-210 promovem a resposta angiogênica em células endoteliais. Objetivo: O presente estudo buscou explorar o efeito do alho e de exercícios físicos voluntários, isoladamente ou em conjunto, nas expressões do miRNA-126 e do miR-210 e na angiogênese cardíaca em ratos com diabetes tipo 1. Métodos: Ratos Wistar machos foram divididos em cinco grupos (n = 7): Controle, Diabetes, Diabetes+Alho, Diabetes+Exercícios e Diabetes+Alho+Exercícios. Introduziu-se diabetes nos animais por estreptozotocina (ip, 50 mg/kg). Os ratos foram então alimentados com homogenato de alho fresco cru (250 mg/kg), ou foram submetidos a exercícios voluntários, ou a uma combinação de alho e exercícios voluntários, durante 6 semanas. As expressões do miRNA-126 e do miRNA-210 no miocárdio foram determinadas por PCR em tempo real, e o perfil lipídico sérico foi medido por kits enzimáticos. A angiogênese foi avaliada por imunocoloração por PECAM-1/CD31 no miocárdio Resultados: O diabetes reduziu a expressão do miRNA-126 cardíaco e da angiogênese (p < 0,05). Por outro lado, houve um aumento da expressão do miRNA-210 no miocárdio dos animais diabéticos (p < 0,001). No entanto, tais efeitos foram revertidos com alho ou exercícios voluntários (p < 0,01). Além disso, o tratamento de ratos diabéticos conjuntamente com alho e exercícios voluntários teve um efeito adicional sobre as expressões do miRNA-126 e do miRNA-210 (p < 0,001). Além disso, tanto os exercícios voluntários quanto o alho melhoraram significativamente os perfis lipídicos séricos (p < 0,001). Conclusões: A indução de diabetes diminuiu a angiogênese no miocárdio, enquanto nosso tratamento com exercícios voluntários de longa duração e alho melhorou a angiogênese miocárdica. Estas alterações devem-se, possivelmente, ao aumento das expressões do miRNA-126 e do miRNA no miocárdio.


Subject(s)
Animals , Male , Physical Conditioning, Animal/physiology , Neovascularization, Physiologic/physiology , Coronary Vessels/physiopathology , MicroRNAs/analysis , Diabetes Mellitus, Type 1/physiopathology , Garlic/chemistry , Triglycerides/blood , Immunohistochemistry , Random Allocation , Cholesterol/blood , Reproducibility of Results , Treatment Outcome , Rats, Wistar , Platelet Endothelial Cell Adhesion Molecule-1/analysis , MicroRNAs/physiology , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/therapy , Real-Time Polymerase Chain Reaction , Heart/physiopathology
6.
São Paulo; s.n; s.n; 2019. 110 p. graf, tab.
Thesis in English | LILACS | ID: biblio-1023378

ABSTRACT

Metabolic Syndrome (MetS) is a combination of diseases interrelated and associated with increased mortality and risk of cardiovascular events. Among the elucidated molecular mechanisms of MetS, there are several genes regulated by miRNAs - small non-coding RNAs. A large number of transcriptomic studies in public databases integrated with new analysis methods can generate new insights. Therefore, this study aimed to identify circulating miRNAs and their target genes in MetS using a Systems Biology approach. For this, we used GEO-NCBI to download and analyse 26 microarray transcriptome studies of MetS and obesity. After preprocessing, the data underwent differential expression (LIMMA method), gene co-expression (CEMiTool), and enrichment (GSEA, Reactome) analyses. We retrieved a gene expression signature for subcutaneous adipose tissue (SAT) for obese individuals that included 291 consistent differentially expressed genes (DEG). This signature had a positive normalized enrichment score (NES) for adaptive immune system activation responses, and negative NES for metabolic pathways. The consensus co-expression network of SAT revealed 3 communities (CM) of densely interconnected genes. These CMs had a high number of up regulated genes and a consistent positive NES among the studies. The co-expressed genes of these 3 CMs were related to neutrophil degranulation, infiltration of immune system cells, and inflammatory processes. Also, a small brazillian cohort (6 individuals with MetS and 6 controls) underwent a seric miRNA profiling using PCR array. From the 222 miRNAs detected in serum, the differential expression analysis identified 4 upregulated miRNAs (miR-30c-5p, miR-421, miR-542-5p and miR-574) in MetS patients (p<0.01). The integrative miRNAs-mRNAs analysis revealed that the circulating upregulated miRNAs had 12 targets in the SAT, 3 targets in the liver; and no targets in the muscle and blood. Many of these target genes are known modulators of proinflammatory pathways. In conclusion, the use of Systems Biology in the analysis of gene networks and circulating miRNAs identified some potential molecular and pathophysiological mechanisms of the Metabolic Syndrome. The circulating miRNAs identified in this study are potential biomarkers and/or therapeutic targets. However, further studies are needed to validate these miRNAs and their target mRNA


A Síndrome Metabólica (MetS) é um conjunto de doenças inter-relacionadas e associadas ao aumento de mortalidade e risco de eventos cardiovasculares. Entre os mecanismos moleculares elucidados da MetS, existem muitos genes regulados por miRNAs - RNAs pequenos não codificadores. O grande número de estudos transcriptômicos em banco dados públicos integrado a novos métodos de análise podem gerar novas descobertas. Deste modo, o objetivo deste estudo foi identificar miRNAs circulantes e genes alvos na MetS usando a abordagem de Biologia de Sistemas. Para isso, GEO-NCBI foi usado para obter e analisar 26 estudos de transcriptoma por microarray de MetS e obesidade. Após o pré-processamento, realizamos análises de expressão diferencial (método LIMMA), co-expressão gênica (CEMiTool), e enriquecimento (GSEA, Reactome). Identificamos uma assinatura de expressão gênica do tecido adiposo subcutâneo (SAT) de indivíduos obesos, composta por 291 genes consistentemente diferencialmente expressos (DEG). Essa assinatura teve um escore de enriquecimento normalizado (NES) positivo para ativação de respostas do sistema imune adaptativo, e NES negativo para vias de metabolismo. A rede consenso de co-expressão do SAT revelou 3 comunidades (CM) de genes densamente interconectadas. Essas CMs continham muitos genes regulados positivamente e com consistência de NES positivo entre os estudos. Os genes co-expressos dessas 3 comunidades pertenciam a vias de a degranulação de neutrófilos, infiltração de células do sistema imune e processos inflamatórios. Além disso, uma pequena coorte brasileira (6 indivíduos com MetS e 6 controles) foi submetida à dosagem sérica de miRNAs por PCR array. Dos 222 miRNAs detectados no soro, a análise de expressão diferencial identificou 4 miRNAs regulados positivamente (miR-30c-5p, miR-421, miR-542-5p e miR-574) nos pacientes com MetS (p<0.01). A análise integrativa miRNAs-mRNAs revelou que osmiRNAs circulantes superexpressos tinham 12 alvos no SAT, 3 alvos no fígado; e nenhum alvo no músculo e no sangue. Muitos desses alvos são moduladores de vias ró-inflamatórias. Em conclusão, a utilização da Biologia de Sistemas na análise de redes gênicas e miRNAs circulantes identificou alguns potenciais mecanismos moleculares e fisiopatológicos da Síndrome Metabólica. Os miRNAs circulantes identificados neste trabalho são potenciais biomarcadores e/ou alvos terapêuticos. Entretanto, mais estudos são necessários para validar esses miRNAs e seus mRNAs alvos


Subject(s)
Metabolic Syndrome/diagnosis , MicroRNAs/analysis , Systems Biology/instrumentation , RNA, Messenger/analysis , Gene Regulatory Networks , Obesity/classification
7.
Braz. j. med. biol. res ; 52(4): e7728, 2019. graf
Article in English | LILACS | ID: biblio-1001506

ABSTRACT

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.


Subject(s)
Humans , Animals , Rats , Pituitary Neoplasms/pathology , Adenoma/pathology , RNA, Long Noncoding/physiology , Enzyme-Linked Immunosorbent Assay , Transfection , Adenoma/genetics , Adenoma/metabolism , Cell Movement/physiology , Cell Survival/physiology , Blotting, Western , Apoptosis/physiology , MicroRNAs/analysis , Cell Line, Tumor , STAT3 Transcription Factor/analysis , Janus Kinase 1/analysis , Janus Kinase 1/metabolism , Cell Migration Assays , Forkhead Box Protein M1/analysis , Forkhead Box Protein M1/metabolism , Luciferases
8.
Braz. j. med. biol. res ; 51(7): e7220, 2018. tab, graf
Article in English | LILACS | ID: biblio-889115

ABSTRACT

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.


Subject(s)
Humans , RNA-Binding Proteins/drug effects , Carcinoma, Hepatocellular/drug therapy , Coumarins/pharmacology , MicroRNAs/drug effects , Liver Neoplasms/drug therapy , Reference Values , Sincalide/analysis , Time Factors , Virus Replication/drug effects , Cell Survival/drug effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , RNA-Binding Proteins/analysis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , MicroRNAs/analysis , Cell Proliferation/drug effects , Hep G2 Cells , Real-Time Polymerase Chain Reaction , Liver Neoplasms/genetics , Liver Neoplasms/virology
9.
Braz. j. med. biol. res ; 51(3): e6329, 2018. graf
Article in English | LILACS | ID: biblio-889035

ABSTRACT

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.


Subject(s)
Animals , Male , Rats , Alcoholism/metabolism , Diabetes Mellitus, Experimental/metabolism , Endothelin-1/analysis , MicroRNAs/analysis , Penis/metabolism , Receptor, Endothelin A/analysis , Receptor, Endothelin B/analysis , Alcoholism/complications , Alcoholism/physiopathology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/physiopathology , Immunohistochemistry , Penis/physiopathology , Rats, Wistar
10.
São Paulo; s.n; s.n; 2018. 127 p. tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-982133

ABSTRACT

INTRODUÇÃO: A obesidade é uma doença multifatorial associada com alto risco de desenvolver resistência à insulina (RI), diabetes melito tipo 2 (DT2), síndrome metabólica (SM) e doenças cardiovasculares (DCV). Os mecanismos moleculares da fisiopatologia das doenças metabólicas não são totalmente compreendidos, emergindo a necessidade de novas estratégias. As modificações epigenéticas, especialmente os miRNAs, podem representar uma importante ferramenta, uma vez que são relacionados com o desenvolvimento de diversas doenças complexas, como as metabólicas. OBJETIVO: Avaliar o perfil de expressão de miRNAs séricos de indivíduos com obesidade, síndrome metabólica e resistência à insulina e sua relação com os mecanismos moleculares envolvidos nas suas fisiopatologias. CASUÍSTICA E MÉTODOS: O estudo foi realizado no Instituto Dante Pazzanese de Cardiologia (IDPC) e no Hospital Universitário da Universidade de São Paulo (HU/USP). Um total de 233 indivíduos foram distribuídos em três grupos: peso normal (n=47), sobrepeso (84) e obesos (102), de acordo com seu índice de massa corpórea (IMC). Os mesmos pacientes também foram re-classificados em com SM (n=89) e sem SM (n=144) de acordo com recomendações da Federação internacional de Diabetes e em com RI (n=123) e sem RI (n=110). As informações biodemográficas e amostras de sangue foram coletadas para testes laboratoriais clínicos e análises de perfis de expressão de miRNA pela PCR array (372 alvos). Analises in silico com a ferramenta Ingenuity Pathway Analysis (IPA) foi realizada para integração do perfil de miRNAs e seus genes alvos com doenças metabólicas. RESULTADOS: Os pacientes obesos e com RI, apresentaram aumento similar das concentrações de marcadores dos perfis lipídico, glicídico e inflamatório quando comparado com indivíduos com peso normal e sem RI. O perfil de miRNAs dos grupos obesos e com RI mostrou que o miR-346 (p=0,010; p=0,019), 483-5p (p=0,034; p=0,020), 296-3p (p=0,010; p=0,008), 1224-3p (p=0,044; p=0,012) e 95-3p (p=0,003; p=0,019) foram expressos similarmente nestes pacientes quando comparados a seus respectivos controles. Estes miRNAs, por predição in silico, desempenham um importante papel na regulação de vias metabólicas associadas a doenças hepáticas gordurosas, inflamação, DCV e doenças metabólicas. Na SM, o grupo portador da doença apresentou altas concentrações de marcadores do perfil lipídico, glicídico e inflamatório. O perfil de expressão de miRNA indicou uma expressão reduzida do miR-183-5p (p=0,008) e miR-301a-3p (p=0,007) e aumentada do miR-542-5p (p=0,003), miR-424-3p (p=0,040), miR-326 (p=0,037), miR-421 (p=0,023) e miR-574-3p (p=0,001) no grupo SM comparado ao sem SM. A análise de regressão linear múltipla stepwise indicou que a expressão aumentada do miR-421 explica em 22,5% o aumento da associação de Hb1Ac, LDL-C, PAI-1, HOMA-IR, glicose e insulina (p <0,001, p=0,014, p=0,009, p <0,001, p=0,001 e p=0,007, respectivamente). Além disso o miR-421 foi predito in silico estar associado com a regulação de 11 mRNA alvos envolvidos em vias regulatórias de doenças como infarto do miocárdio, obesidade, diabetes melito, esteatose hepática, tolerância a glicose, distúrbio do metabolismo da glicose, hiperglicemia e resistência à insulina, bem como atua em vias regulatórias da resposta inflamatória, sendo associado com a variabilidade na quantidade de citocinas, massa do tecido adiposo e quantidade de leptina no sangue. Mediante a relação do miR-421 com a homeostase da glicose, sua expressão foi testada em pacientes com RI e interessantemente se manteve aumentada nesses pacientes em comparação com os sem RI (p=0.014). CONCLUSÕES: Os miR-346, miR-483-5p, miR-296-3p, miR-1224-3p e miR-95-3p podem ser considerados potenciais biomarcadores como preditores de DCV em pacientes obesos e resistência à insulina e o miR-421 um potencial biomarcador precoce de predição de risco de DCV em pacientes com síndrome metabólica


BACKGROUND: Obesity is a multifactorial disease associated with high risk of developing insulin resistance (IR), Diabetes mellitus type 2 (DT2), metabolic syndrome (MetS) and cardiovascular disease (CVD). Molecular mechanisms of metabolic diseases' pathophysiology are not fully understood, emerging the need for new strategies. Epigenetic modifications, especially miRNAs, may represent an important tool, since they are related to the development of several complex diseases, such as those metabolic. OBJECTIVE: Evaluate miRNA-derived serum expression profile in individuals with obesity, MetS and IR and their relationship with molecular mechanisms involved in the metabolic disease pathophysiology. MATERIAL AND METHODS: The study was conducted at the Dante Pazzanese Institute of Cardiology (IDPC) and at University Hospital of the University of São Paulo (HU/USP). Two hundred thirty-three subjects were distributed into three groups: normal weight (n = 47), overweight (n=84) and obese (n=102), according to their body mass index (BMI). The same patients were also re-classified as MetS (n=89) and Non-MetS (n=144) according to recommendations of the International Diabetes Federation and in IR (n=123) and Non-IR. Biodemographic data were registered and blood samples were collected for clinical laboratory tests and miRNA expression profiles by PCR array (372 targets). In silico analysis using Ingenuity Pathway Analysis (IPA) tool was performed to integrate the miRNA profile and their target genes with metabolic diseases. RESULTS: Obese and IR patients had similar increases in the concentrations of lipid, glucose and inflammatory markers when compared to individuals with normal weight and without IR, respectively. miRNA profile of obese and IR groups showed that miR-346 (p = 0.010, p = 0.019), 483-5p (p = 0.034, p = 0.020), 296-3p (p = 0.010, p = 0.008), 1224-3p (p = 0.044, p = 0.012) and 95-3p (p = 0.003, p = 0.019) were similarly expressed in these patients in comparison to their respective controls. These miRNAs, by in silico prediction, play an important role in the regulation of metabolic pathways associated with fatty liver diseases, inflammation, CVD and metabolic diseases. In MetS, patients diagnosed with the disease showed high concentrations of lipid, glycogen and inflammatory markers. The miRNA expression profile indicated a reduced expression of miR-183-5p (p=0.008) and miR-301a-3p (p=0.007) and increased miR-542-5p (p=0.003), miR- 3 (p=0.040), miR-326 (p=0.037), miR-421 (p=0.023) and miR-574-3p (p=0.001) in the MetS group compared to Non-MetS. Stepwise multiple linear regression analysis indicated that increased expression of miR-421 explain in 22.5% increased of Hb1Ac, LDL-C, PAI-1, HOMA-IR, glucose and insulin (p<0.001, p=0.014, p=0.009, p<0.001, p=0.001 and p=0.007, respectively). In addition the miR-421 was predicted in silico to associated with regulation of eleven mRNA of gene targets involved in regulatory pathways of diseases such as myocardial infarction, obesity, diabetes mellitus, hepatic steatosis, glucose tolerance, glucose metabolism disorder, hyperglycemia and insulin resistance, as well inflammatory response and is also associated with variability in the amount of cytokines, adipose tissue mass and amount of leptin in the blood. Due the relationship of miR-421 with glucose homeostasis, the miRNA expression was tested in patients with IR. Interestingly, miR-421 remained increased in these patients compared to those without IR (p = 0.014). CONCLUSIONS: The miR-346, miR-483-5p, miR-296-3p, miR-1224-3p and miR-95-3p may be potential biomarkers to predict CVD in obese and IR patients, while the miR-421 is a potential biomarker to predict CVD in MetS patients


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Insulin Resistance/genetics , Cardiovascular Diseases/diagnosis , MicroRNAs/analysis , Obesity/complications , Computer Simulation/statistics & numerical data , Biomarkers/analysis , Metabolic Syndrome , Glycemic Index
11.
Braz. oral res. (Online) ; 32: e126, 2018. graf
Article in English | LILACS | ID: biblio-974437

ABSTRACT

Abstract: Novel biomarkers for screening, diagnosis and monitoring the treatment of nasopharyngeal carcinoma (NPC), one of the most common cancers in Vietnam, are urgently required. Increasing evidence suggests that microRNA-141 (miR-141) is associated with NPC, owing to its ability to affect the expression of genes that modulate tumorigenesis. Unfortunately, research on miR-141 expression in Vietnamese patients is limited. Therefore, the objective of the current study was to evaluate miR-141 expression and assess whether miR-141 might be a potential biomarker for diagnosis of NPC in Vietnamese patients. Total RNA isolated from 40 NPC biopsy samples and 37 non-cancerous samples was analyzed by quantitative reverse-transcription PCR. The miR-141 expression levels were compared between NPC biopsy and non-cancerous samples. The frequency of miR-141 detection was 37.50% and 10.80% in the NPC and non-cancerous samples, respectively (p = 0.0143). The miR-141 expression was 5.27 times higher in tumor samples than non-cancerous samples. Additionally, the RR (Relative risk) and OR (Odds ratio) were 1.83 (95%CI = 1.2576-2.6675, p = 0.0016) and 4.95 (95%CI = 1.4625-16.7541, p = 0.01), respectively. In conclusion, miR-141 was up-regulated in the biopsy samples and thus may be a potential biomarker for NPC in the Vietnamese population.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Young Adult , Nasopharyngeal Neoplasms/genetics , MicroRNAs/analysis , Nasopharyngeal Carcinoma/genetics , Reference Values , Vietnam , Biomarkers, Tumor/analysis , Case-Control Studies , Up-Regulation , Nasopharyngeal Neoplasms/pathology , Asian Continental Ancestry Group , Real-Time Polymerase Chain Reaction , Nasopharyngeal Carcinoma/pathology , Middle Aged
12.
Biol. Res ; 51: 56, 2018. graf
Article in English | LILACS | ID: biblio-1011400

ABSTRACT

BACKGROUND: Glioma is the most prevalent malignant tumor in human central nervous systems. Recently, the development of resistance to radiotherapy in glioma patients markedly vitiates the therapy outcome. MiR-153-3p has been reported to be closely correlated with tumor progression, but its effect and molecular mechanism underlying radioresistance remains unclear in glioma. METHODS: The expression of miR-153-3p was determined in radioresistant glioma clinical specimens as well as glioma cell lines exposed to irradiation (IR) using quantitative real-time PCR. Cell viability, proliferation and apoptosis were then evaluated by MTT assay, colony formation assay, Flow cytometry analysis and caspase-3 activity assay in glioma cells (U87 and U251). Tumor forming was evaluated by nude mice model in vivo. TUNEL staining was used to detect cell apoptosis in nude mice model. The target genes of miR-153-3p were predicted and validated using integrated bioinformatics analysis and a luciferase reporter assay. RESULTS: Here, we found that miR-153-3p was down-regulated in radioresistant glioma clinical specimens as well as glioma cell lines (U87 and U251) exposed to IR. Enhanced expression of miR-153-3p promoted the radiosensitivity, promoted apoptosis and elevated caspase-3 activity in glioma cells in vitro, as well as the radiosensitivity in U251 cell mouse xenografs in vivo. Mechanically, B cell lymphoma-2 gene (BCL2) was identified as the direct and functional target of miR-153-3p. Moreover, restoration of BCL2 expression reversed miR-153-3p-induced increase of radiosensitivity, apoptosis and caspase-3 activity in U251 cells in vitro. In addition, clinical data indicated that the expression of miR-153-3p was significantly negatively associated with BCL2 in radioresistance of glioma samples. CONCLUSIONS: Our findings suggest that miR-153-3p is a potential target to enhance the effect of radiosensitivity on glioma cells, thus representing a new potential therapeutic target for glioma.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Radiation Tolerance/genetics , Genes, bcl-2/physiology , MicroRNAs/radiation effects , MicroRNAs/physiology , Glioma/genetics , Time Factors , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell Survival/radiation effects , Blotting, Western , Analysis of Variance , Gene Targeting/methods , Genes, bcl-2/radiation effects , In Situ Nick-End Labeling , MicroRNAs/analysis , Cell Line, Tumor , Cell Proliferation/radiation effects , Caspase 3/analysis , Real-Time Polymerase Chain Reaction , Flow Cytometry , Glioma/radiotherapy
13.
Braz. j. med. biol. res ; 51(12): e7665, 2018. graf
Article in English | LILACS | ID: biblio-974250

ABSTRACT

Osteosarcoma (OS) has a high incidence, malignity, and frequency of recurrence and metastasis. In this study, we aimed to explore the potential anti-cancer effects of Astragalus polysaccharides (APS) on human OS MG63 cells as well as underlying mechanisms. Viability of MG63 cells was assessed by CCK-8 assay to determine the adequate concentration of APS. Then, effects of APS on MG63 cell proliferation, cell cycle distribution, apoptosis, and migration and invasion were analyzed by BrdU incorporation, PI staining, flow cytometry, and transwell assays, respectively. The expression levels of proteins involved in these physiological processes were assessed by western blot analysis. Afterwards, miR-133a level in APS-treated cells was determined by qRT-PCR, and whether APS affected MG63 cells through regulation of miR-133a was determined. Finally, the activation of c-Jun N-terminal protein kinase (JNK) pathway was detected. We found that APS treatment suppressed the viability, proliferation, migration, and invasion of MG63 cells, as well as induced cell apoptosis. Moreover, APS enhanced the expression of miR-133a in MG63 cells. Knockdown of miR-133a reversed the APS treatment-induced MG63 cell proliferation, migration and invasion inhibition, as well as cell apoptosis. Furthermore, APS inactivated JNK pathway in MG63 cells. Knockdown of miR-133a reversed the APS treatment-induced inactivation of JNK pathway in MG63 cells. To conclude, APS repressed proliferation, migration, and invasion while induced apoptosis of OS MG63 cells by up-regulating miR-133a and then inactivating JNK pathway.


Subject(s)
Humans , Bone Neoplasms/pathology , Cell Movement/drug effects , Apoptosis/drug effects , Astragalus Plant/chemistry , Cell Proliferation/drug effects , Bone Neoplasms/drug therapy , Cell Cycle/drug effects , Up-Regulation/drug effects , Cell Survival/drug effects , Blotting, Western , Reproducibility of Results , Analysis of Variance , MicroRNAs/analysis , Cell Line, Tumor , JNK Mitogen-Activated Protein Kinases/analysis , Antineoplastic Agents/pharmacology
14.
Braz. j. otorhinolaryngol. (Impr.) ; 83(3): 276-284, May-June 2017. tab, graf
Article in English | LILACS | ID: biblio-889263

ABSTRACT

Abstract Introduction: Salivary gland tumors (SGTs) are rare head and neck malignancies consisting of a spectrum of tumors with different biological behaviors. Objective: In this study we aimed to find out differential expression of microRNA profiles between benign and malignant SGTs. Methods: We investigated the possible role of 95 microRNAs in the 20 patients with salivary gland tumors with comparison of 17 patients without malignancy or salivary gland diseases. Sixteen of the tumors were benign (seven pleomorphic adenomas, nine Warthin tumors), four of them were malignant (two squamous cell carcinomas, one high grade mucoepidermoid carcinoma, one adenocarcinoma). Serum and saliva samples were collected from both patients and control group. Tissue samples of tumor masses were also collected from patient group. Results: Among studied microRNAs miR-21, miR-23a, miR-27a, miR-223, miR-125b, miR-126, miR-146a, miR-30e were down regulated in the benign group compared to control group in the serum samples (p-values are 0.04, 0.00005, 0.00005, 0.0022, 0.031, 0.00008, 0.044, and 0.0007, respectively). When tissue samples were studied miR-21, miR-31, miR-199a-5p, miR-146b, miR-345 were up-regulated in the malignant group compared to benign group (p values are 0.006, 0.02, 0.013, 0.013, 0.041, respectively). miR-30e showed statistically significant up-regulation in malignant tumor group's plasma samples compared to benign group (p = 0.034). There was no statistically significant difference in saliva samples between groups. Conclusion: Our results showed that different microRNAs may play role in salivary tumor pathogenesis according to biological behavior. Although there was no difference in saliva samples between groups, according to tissue and serum samples miR-21 and 30e may have an important role; since they were down-regulated in benign tumors whereas up-regulated in malignant ones.


Resumo Introdução: Os tumores da glândula salivar (TGS) são lesões malignas raras de cabeça e pescoçoque consistem em um espectro de tumores com diferentes comportamentos biológicos. Objetivo: Neste estudo, tivemos como objetivo identificar a expressão diferencial de perfis demicroRNA entre TGS benignos e malignos. Método: Investigamos a possível participação de 95 microRNA em 20 pacientes com tumoresde glândulas salivares comparados com 17 pacientes sem doença maligna ou doenças das glân-dulas salivares; 16 dos tumores eram benignos (sete adenomas pleomórficos, nove tumores deWarthin), quatro eram malignos (dois carcinomas espinocelulares, carcinoma mucoepidermoidede alto grau, um adenocarcinoma). As amostras de soro e saliva foram coletadas de pacien-tes e do grupo controle. Amostras de tecido dos tumores também foram colhidas do grupo depacientes com tumores. Resultados: Entre os microRNA estudados, miR-21, miR-23a, miR-27a, miR-223, miR-125b, miR-126, miR-146a, miR-30e foram infrarregulados no grupo benigno em comparação com o grupocontrole nas amostras do soro (os valores de p são 0,04, 0,00005, 0,00005, 0,0022, 0,031,0,00008, 0,044 e 0,0007, respectivamente). Quando as amostras de tecido foram estudadas,miR-21, o miR-31, o miR-199-5p, miR-146b, o miR-345 foram suprarregulados no grupo malignoem relação ao grupo benigno (valores de p são 0,006, 0,02, 0,013, 0,013, 0,041, respectiva-mente). O miR-30e apresentou suprarregulação estatisticamente significativa em amostras deplasma do grupo de tumor maligno em relação ao grupo benigno (p = 0,034). Não houve diferençaestatisticamente significativa em amostras de saliva entre os grupos. Conclusão: Nossos resultados mostraram que diferentes microRNA podem desempenhar umpapel na patogenia do tumor salivar de acordo com o comportamento biológico. Embora nãotenha havido diferença em amostras de saliva entre os grupos, de acordo com as amostras detecido e de soro, miR-21 e 30e podem ter um papel importante, já que foram infrarreguladosnos tumores benignos enquanto suprarregulados nos tumores malignos.


Subject(s)
Humans , Male , Female , Salivary Gland Neoplasms/diagnosis , MicroRNAs/analysis , Saliva/chemistry , Salivary Gland Neoplasms/genetics , Biomarkers, Tumor/analysis , Case-Control Studies , Gene Expression Regulation, Neoplastic/genetics , Prospective Studies
15.
Acta cir. bras ; 32(3): 243-250, Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-837691

ABSTRACT

Abstract Purpose: To evaluate the expression of EGFR, KRAS genes, microRNAs-21 and 203 in colon and rectal cancer samples, correlated with their age at diagnosis, histological subtype, value of pretreatment CEA, TNM staging and clinical outcome. Methods: Expression of genes and microRNAs by real time PCR in tumor and non-tumor samples obtained from surgical treatment of 50 patients. Results: An increased expression of microRNAs-21 and 203 in tumor samples in relation to non-tumor samples was found. There was no statistically significant difference between the expression of these genes and microRNAs when compared to age at diagnosis and histological subtype. The EGFR gene showed higher expression in relation to the value of CEA diagnosis. The expression of microRNA-203 was progressively lower in relation to the TNM staging and was higher in the patient group in clinical remission. Conclusions: The therapy of colon and rectum tumors based on microRNAs remains under investigation reserving huge potential for future applications and clinical interventions in conjunction with existing therapies. We expect, based on the exposed data, to stimulate the development of new therapeutic possibilities, making the treatment of these tumors more effective.


Subject(s)
Humans , Male , Female , Middle Aged , Aged , Aged, 80 and over , Colorectal Neoplasms/genetics , Adenocarcinoma/genetics , Gene Expression , Proto-Oncogene Proteins p21(ras)/analysis , Genes, ras , Genes, erbB-1 , MicroRNAs/analysis , Colorectal Neoplasms/pathology , Colorectal Neoplasms/drug therapy , Adenocarcinoma/pathology , Adenocarcinoma/drug therapy , Carcinoembryonic Antigen/analysis , Biomarkers, Tumor/analysis , Prospective Studies , Age Factors , Treatment Outcome , Real-Time Polymerase Chain Reaction , Neoplasm Staging
16.
Acta cir. bras ; 32(1): 38-45, Jan. 2017. graf
Article in English | LILACS | ID: biblio-837673

ABSTRACT

Abstract Purpose: To evaluate the expression of endothelial and inducible NOS in addition to the miRNA-27b in the corpus cavernosum and peripheral blood of healthy rats, diabetic rats, alcoholic rats and rats with both pathologies. Methods: Forty eight Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D) and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study protein expressions of eNOS and iNOS by immunohistochemistry and expression of miRNA-27b in the corpus cavernosum and peripheral blood. Results: Immunohistochemistry for eNOS and iNOS showed an increase in cavernosal smooth muscle cells in the alcoholic, diabetic and alcoholic-diabetic groups when compared with the control group. Similarly, the mRNA levels for eNOS were increased in cavernosal smooth muscle (CSM) in the alcoholic, diabetic and alcoholic-diabetic groups and miRNA-27b were decreased in CSM in the alcoholic, diabetic and alcoholic-diabetic groups. Conclusion: The major new finding of our study was an impairment of relaxation of cavernosal smooth muscle in alcoholic, diabetic, and alcoholic-diabetic rats that involved a decrease in the nitric oxide pathway by endothelium-dependent mechanisms accompanied by a change in the corpus cavernosum contractile sensitivity.


Subject(s)
Animals , Male , Rats , Penis/chemistry , MicroRNAs/analysis , Diabetes Mellitus, Experimental/metabolism , Alcoholism/metabolism , Nitric Oxide Synthase Type II/analysis , Nitric Oxide Synthase Type III/analysis , Penis/physiopathology , Immunohistochemistry , Rats, Wistar , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/physiopathology , Alcoholism/complications , Alcoholism/physiopathology
17.
Braz. j. med. biol. res ; 50(5): e6359, 2017. graf
Article in English | LILACS | ID: biblio-839294

ABSTRACT

MicroRNAs (miRNAs) play an important role in drug resistance and modulate the efficiency of chemotherapy. A recent study indicated that miR-340 functions as a tumor suppressor in various types of cancer. However, the role of miR-340 in chemotherapy has not been reported yet. In this study, we found that miR-340 enhanced cisplatin (CDDP)-induced cell death. Induction of miR-340-5p expression decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells. Moreover, miR-340-5p decreased the accumulation of MRP1 and MDR1. We further explored the mechanism underlying the promoting effects of miR-340-5p on CDDP-induced cell death. We identified a potential target of miR-340 in the 3′ untranslated region of lysophosphatidic acid acyltransferase (LPAATβ) using the online program Targetscan (http://www.microrna.org). Luciferase reporter assays showed that miR-340 binds to the 3′UTR of LPAATβ. Enforced expression of miR-340-5p decreased the accumulation of LPAATβ in both MG-63 and Saos-2 cells. Silencing LPAATβ decreased the IC50 of CDDP and increased the apoptosis of CDDP-resistant MG-63 and Saos-2 cells, which is consistent with the effect of miR-340-5p on CDDP-induced cell death. Moreover, induced expression of LPAATβ compromised the effects of miR-340-5p on CDDP-induced cell death and accumulation of MRP1 and MDR1. Taken together, our data indicated that miR-340-5p enhanced the sensitivity to CDDP by targeting LPAATβ.


Subject(s)
Humans , Acyltransferases/physiology , Antineoplastic Agents/pharmacology , Bone Neoplasms/drug therapy , Cisplatin/pharmacology , Drug Resistance, Neoplasm/physiology , MicroRNAs/physiology , Osteosarcoma/drug therapy , Acyltransferases/analysis , Acyltransferases/drug effects , Apoptosis/drug effects , Blotting, Western , Bone Neoplasms/physiopathology , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation , Drug Resistance, Neoplasm/drug effects , Luciferases , MicroRNAs/analysis , MicroRNAs/drug effects , Osteosarcoma/physiopathology , Real-Time Polymerase Chain Reaction
18.
Braz. j. med. biol. res ; 50(11): e6246, 2017. graf
Article in English | LILACS | ID: biblio-888947

ABSTRACT

This study investigated the expression and regulation of IL-6R in hepatitis B-associated moderate hepatic fibrosis and cirrhosis. Liver tissues, peripheral blood monocytes (PBMs) and serum were collected from 26 hepatitis B patients with liver fibrosis and 35 hepatitis B patients with liver cirrhosis. The levels of Il-6r mRNA expression in these samples were examined by quantitative real-time PCR and IL-6R protein levels were analyzed by western blot and ELISA. MiRNAs that regulate IL-6R expression were predicted by bioinformatics analysis, and validated by dual luciferase reporter assay. Compared with the hepatic fibrosis group, IL-6R was significantly upregulated at both mRNA and protein levels in liver tissues, PBMs and serum samples from the hepatic cirrhosis group (P<0.05). The 3′UTR of Il-6r mRNA was predicted to contain a miR-30b binding site and IL-6R was identified as a possible target of miR-30b. MiR-30b expression was significantly downregulated in samples from hepatic cirrhosis patients compared with hepatic fibrosis patients (P<0.05). In conclusion, IL-6R was upregulated while miR-30b was decreased in patients with liver cirrhosis. The miR-30 can directly regulate the expression of IL-6R.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Hepatitis B/metabolism , Liver Cirrhosis/metabolism , MicroRNAs/metabolism , Receptors, Interleukin-6/metabolism , Down-Regulation , Hepatitis B/blood , Liver Cirrhosis/blood , MicroRNAs/analysis , MicroRNAs/chemistry , Receptors, Interleukin-6/analysis , Reference Values , Time Factors , Up-Regulation
19.
São Paulo; s.n; s.n; 2016. 134 p +anexos tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-846592

ABSTRACT

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel


The antiplatelet therapy is often indicated for the prevention and treatment of cardiovascular diseases. Dual antiplatelet therapy with clopidogrel and acetylsalicylic acid (ASA) has often been adopted in patients with coronary artery disease (CAD), but it has been ineffective in a significant portion of the population with genotype of responders. This fact leads us to question whether other molecular mechanisms may be influencing the response to these drugs. Recent studies suggest that small non-coding RNA sequences known as microRNAs (miRNAs) may be closely related to response to drug-therapeutic treatment, controlling proteins involved in pharmacokinetics and pharmacodynamics. The aim of this study was to evaluate the profile of miRNAs in whole blood, looking to better clarify mechanisms involved in ASA and clopidogrel response. For this purpose, we selected CAD patients who showed different responses to dual antiplatelet therapy determined by aggregation test. Based on the phenotypes, the miRNA expression profiles were compared between the platelet aggregation values categorized into tertiles (T) of response. The T1 group consisted of responding patients, the T2 consisted of intermediate responders and the T3 consisted of non-responders. The miRNA profiles were obtained after next-generation sequencing and data were analyzed by Deseq2 package. Results showed that 18 miRNAs were differentially expressed between the two extreme tertiles. By Ingenuity® software prediction analysis, 10 miRNAs showed important targets related with activation and aggregation of blood platelets. Of the 10 miRNAs, 4 of them, which were down-expressed on sequencing, showed the same fold-regulation when expression profiles were analyzed by quantitative polymerase chain reaction (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR-30a-5p and has-hsa- let-7g-5p. By target prediction analysis, it was observed that, when the four miRNAs are simultaneously down-expressed, they predict activation of platelet aggregation. Furthermore, hsa-miR-423-5p, hsa-miR-744-5p, and hsa-let-7g-5p showed correlation with the lipid profile of patients which, in turn, demonstrated influence in aggregation values reaching T3 of response to both drugs. Therefore, we concluded that increased platelet aggregation rates may be indirectly related to the expression profiles of hsa-miR-423-3p, hsa-miR-744-5p and hsa-let-7g-5p. It is suggested that the evaluation of the expression profile of these three miRNAs in the peripheral blood of patients with CAD may predict inadequate therapeutic response to aspirin and clopidogrel


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , MicroRNAs/analysis , Platelet Aggregation Inhibitors , Aspirin/administration & dosage , Cardiovascular Diseases , Coronary Vessels , Molecular Biology , Pharmacogenetics
20.
São Paulo; s.n; s.n; 2016. 134 p. graf, ilus, tab.
Thesis in Portuguese | LILACS | ID: biblio-881509

ABSTRACT

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel


The antiplatelet therapy is often indicated for the prevention and treatment of cardiovascular diseases. Dual antiplatelet therapy with clopidogrel and acetylsalicylic acid (ASA) has often been adopted in patients with coronary artery disease (CAD), but it has been ineffective in a significant portion of the population with genotype of responders. This fact leads us to question whether other molecular mechanisms may be influencing the response to these drugs. Recent studies suggest that small non-coding RNA sequences known as microRNAs (miRNAs) may be closely related to response to drug-therapeutic treatment, controlling proteins involved in pharmacokinetics and pharmacodynamics. The aim of this study was to evaluate the profile of miRNAs in whole blood, looking to better clarify mechanisms involved in ASA and clopidogrel response. For this purpose, we selected CAD patients who showed different responses to dual antiplatelet therapy determined by aggregation test. Based on the phenotypes, the miRNA expression profiles were compared between the platelet aggregation values categorized into tertiles (T) of response. The T1 group consisted of responding patients, the T2 consisted of intermediate responders and the T3 consisted of non-responders. The miRNA profiles were obtained after next-generation sequencing and data were analyzed by Deseq2 package. Results showed that 18 miRNAs were differentially expressed between the two extreme tertiles. By Ingenuity® software prediction analysis, 10 miRNAs showed important targets related with activation and aggregation of blood platelets. Of the 10 miRNAs, 4 of them, which were down-expressed on sequencing, showed the same fold-regulation when expression profiles were analyzed by quantitative polymerase chain reaction (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR-30a-5p and has-hsa- let-7g-5p. By target prediction analysis, it was observed that, when the four miRNAs are simultaneously down-expressed, they predict activation of platelet aggregation. Furthermore, hsa-miR-423-5p, hsa-miR-744-5p, and hsa-let-7g-5p showed correlation with the lipid profile of patients which, in turn, demonstrated influence in aggregation values reaching T3 of response to both drugs. Therefore, we concluded that increased platelet aggregation rates may be indirectly related to the expression profiles of hsa-miR-423-3p, hsa-miR-744-5p and hsa-let-7g-5p. It is suggested that the evaluation of the expression profile of these three miRNAs in the peripheral blood of patients with CAD may predict inadequate therapeutic response to aspirin and clopidogrel


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Aspirin/pharmacology , Coronary Artery Disease/pathology , MicroRNAs/analysis , Platelet Aggregation Inhibitors/pharmacology , Biological Specimen Banks/statistics & numerical data , Gene Library , Polymerase Chain Reaction/methods
SELECTION OF CITATIONS
SEARCH DETAIL