Expressão de microRNAs em pacientes com diagnóstico histopatológico prévio de Displasia Neuronal Intestinal do Tipo B
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Data
2018-05-29
Autores
Angelini, Marcos Curcio
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Universidade Estadual Paulista (Unesp)
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Introdução: A Displasia Neuronal Intestinal tipo B (DNI-B) é uma entidade patológica caracterizada por anormalidades nos plexos nervosos submucosos do sistema nervoso entérico, comumente associada à constipação intestinal na infância. Os microRNAs (miRNAs) são pequenos RNAs não-codificantes com um papel importante na regulação da expressão gênica, e têm sido considerados potenciais biomarcadores diagnósticos em diversas doenças. Objetivo: Determinar o perfil de expressão global de miRNAs em amostras de plasma e tecido de biópsia retal de pacientes com DNI-B. Métodos: Cinqüenta pacientes, com idade entre 0 e 15 anos, diagnosticados com DNI-B entre 1998 a 2010, foram incluídos no estudo. Amostras de sangue periférico foram coletadas de todos os 50 pacientes e 10 crianças saudáveis, sem sintomas intestinais (grupo controle). Dos 50 pacientes dos quais o sangue foi coletado, 29 também apresentavam fragmentos embolcados de tecido retal suficiente para extração de RNA. Estes materiais foram processados por microdissecção e agrupados de acordo com as diferentes camadas da parede intestinal. A extração de miRNA foi realizada utilizando o kit miRNeasy Serum / Plasma e o kit de recuperação de todos os Nucleic Acid Isolation para tecidos de biópsia. A análise global da expressão de miRNA foi realizada utilizando a plataforma TaqMan Array Human MicroRNA Card A. Análise de correlação entre a expressão de miRNA em amostras de plasma e biópsia, bem como entre os tecidos derivados das camadas intestinais distintas foi realizada. Resultados: o miRNA let-7a foi superexpresso (~ 7 vezes mais) no plasma de pacientes com DNI-B em comparação com os controles. A análise da expressão do miRNA em amostras de biópsia utilizou a camada mucosa como controle, uma vez que as células ganglionares do plexo nervoso, que estão alteradas em pacientes com DNI-B, não estão localizadas nessa camada. Os miRNAs que apresentaram expressão aumentada (miR-146a, miR-146b) ou diminuída (miR-451, miR-99a, miR-130a, miR-374-5p, miR-100, miR-365, miR-145, miR133b), simultaneamente na submucosa e na associação entre as camadas submucosa e muscular, pode ter atividade relacionada aos neurônios do sistema nervoso entérico. O miR-451 foi o único miRNA significativamente desregulado em tecidos de plasma e nos fragmentos de reto dos pacientes. O miR-451 desempenha um papel regulador nas vias de proliferação celular e apoptose e pode estar ligado à regulação da síntese proteica de GNA11, que por sua vez atua na modulação do sistema transmembrana, incluindo contração muscular intestinal. Conclusão: Nossos resultados demonstram a presença de alterações de miRNA na submucosa e na camada muscular do reto, bem como no plasma de pacientes com DNI-B.
Introduction: Intestinal Neuronal Dysplasia type B (IND-B) is a pathological entity characterized by abnormalities in the submucous nerve plexuses of the enteric nervous system, commonly associated with intestinal constipation in childhood. microRNAs (miRNAs) are small non-coding RNAs with a potent role in gene expression regulation, and have been demonstrated as potential diagnostic biomarkers in several diseases. Objective: To determine the global expression profile of miRNAs in plasma and rectal biopsy tissue samples from patients with IND-B. Methods: Fifty patients, aged 0 to 15 years, who had been diagnosed with IND-B, from 1998 to 2010, were included in the study. Peripheral blood samples were collected from all 50 patients and an additional 10 healthy children, without intestinal symptoms (control group). Of the 50 patients from whom blood was collected, 29 also had sufficient rectal tissue for RNA extraction. Rectal biopsies were processed by microdissection and pooled according the different layers of the intestinal wall. miRNA extraction was performed using the miRNeasy Serum/Plasma Kit and the Recover All Total Nucleic Acid Isolation kit for biopsy tissues. Global miRNA expression analysis was performed using the TaqMan Array Human MicroRNA Card A platform. Correlation analysis between miRNA expression in plasma and biopsy samples as well as among tissues derived from the distinct intestinal layers was performed. Results: miRNA let-7a was over-expressed (~7 fold higher) in plasma from patients with IND-B compared to controls. miRNA expression analysis in biopsy samples used the mucosal layer as reference control, since ganglion cells of the nerve plexus, which are mostly affected in IND-B patients, are not located in this layer. miRNAs that showed increased (miR-146a, miR-146b) or decreased (miR-451, miR-99a, miR-130a, miR-374-5p, miR-100, miR-365, miR-145, miR 133b) expression, simultaneously in the submucosa and in the association between the submucosal and muscular layers, may have activity related to the neurons of the enteric nervous system. miR-451 was the only miRNA significantly deregulated in patient plasma and biopsy tissues.miR-451 plays a regulatory role on cell proliferation and apoptosis pathways and may be linked to the regulation of GNA11 protein synthesis, which in turn acts on the modulation of transmembrane systems, including intestinal muscle contraction. Conclusion: Our results demonstrate the presence of miRNA alterations in the submucosa and muscular layers of the rectum, as well as in plasma from patients with IND-B.
Introduction: Intestinal Neuronal Dysplasia type B (IND-B) is a pathological entity characterized by abnormalities in the submucous nerve plexuses of the enteric nervous system, commonly associated with intestinal constipation in childhood. microRNAs (miRNAs) are small non-coding RNAs with a potent role in gene expression regulation, and have been demonstrated as potential diagnostic biomarkers in several diseases. Objective: To determine the global expression profile of miRNAs in plasma and rectal biopsy tissue samples from patients with IND-B. Methods: Fifty patients, aged 0 to 15 years, who had been diagnosed with IND-B, from 1998 to 2010, were included in the study. Peripheral blood samples were collected from all 50 patients and an additional 10 healthy children, without intestinal symptoms (control group). Of the 50 patients from whom blood was collected, 29 also had sufficient rectal tissue for RNA extraction. Rectal biopsies were processed by microdissection and pooled according the different layers of the intestinal wall. miRNA extraction was performed using the miRNeasy Serum/Plasma Kit and the Recover All Total Nucleic Acid Isolation kit for biopsy tissues. Global miRNA expression analysis was performed using the TaqMan Array Human MicroRNA Card A platform. Correlation analysis between miRNA expression in plasma and biopsy samples as well as among tissues derived from the distinct intestinal layers was performed. Results: miRNA let-7a was over-expressed (~7 fold higher) in plasma from patients with IND-B compared to controls. miRNA expression analysis in biopsy samples used the mucosal layer as reference control, since ganglion cells of the nerve plexus, which are mostly affected in IND-B patients, are not located in this layer. miRNAs that showed increased (miR-146a, miR-146b) or decreased (miR-451, miR-99a, miR-130a, miR-374-5p, miR-100, miR-365, miR-145, miR 133b) expression, simultaneously in the submucosa and in the association between the submucosal and muscular layers, may have activity related to the neurons of the enteric nervous system. miR-451 was the only miRNA significantly deregulated in patient plasma and biopsy tissues.miR-451 plays a regulatory role on cell proliferation and apoptosis pathways and may be linked to the regulation of GNA11 protein synthesis, which in turn acts on the modulation of transmembrane systems, including intestinal muscle contraction. Conclusion: Our results demonstrate the presence of miRNA alterations in the submucosa and muscular layers of the rectum, as well as in plasma from patients with IND-B.
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Palavras-chave
Displasia Neuronal Intestinal, MicroRNA, Disganglionoses intestinais, Constipação intestinal, Intestinal Neuronal Dysplasia, intestinal constipation