Perfil de expressão de microRNAs no esôfago de crianças portadoras de estenose cáustica
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Data
2018-05-29
Autores
Oliveira Junior, Wilson Elias de
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Universidade Estadual Paulista (Unesp)
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Introdução: Por volta de oitenta por cento dos acidentes envolvendo ingestão de cáusticos ocorrem em crianças. A estenose cáustica do esôfago é a sua principal complicação com grande morbidade. Lesões neoplásicas esofágicas podem desenvolver-se como uma complicação tardia desta estenose com um tempo médio de aparecimento entre o acidente e o desenvolvimento neoplásico de 15 a 30 anos. Considerando este risco, biopsias seriadas do esôfago são recomendadas com o objetivo de detecção precoce de displasias. Assim, um conhecimento abrangente da relação biológica entre cáusticos e neoplasia esofágica é de grande importância na identificação de novos biomarcadores que possibilitariam tratamento precoce. MicroRNAs (miRNAs) são RNAs pequenos, não codificadores de proteínas que regulam importantes processos celulares e têm se mostrado como robustos biomarcadores. O perfil global de expressão de miRNAs nesta população, seguida da identificação dos miRNA-alvo, pode levar à identificação da presença e magnitude do dano ao material genético em amostra de tecido esofágico obtido de pacientes portadores de estenose cáustica. Objetivos: Determinar o perfil global da expressão de miRNAs em células da mucosa esofágica de crianças portadoras de lesões por ingestão de cáusticos, com o objetivo de identificar miRNAs como biomarcadores associados a tumorigênese esofágica nesta população específica. Materiais e Métodos: Vinte e sete amostras esofágicas fixadas em formalina e embebidas em parafina (FFPE) de 15 pacientes foram divididas em dois grupos de acordo com o tempo transcorrido entre o acidente e a estenose (Grupo A: menos de 5 anos; Grupo B: mais de 5 anos). Estes tecidos foram então pareados de acordo com o gênero, idade e ano da biópsia, e comparados a amostras esofágicas normais (macro e microscopicamente). Após o pareamento, as amostras foram microdissecadas com agulha e submetidas a extração do RNA usando o Recover AllTM Total Nucleic Acid Isolation Kit (Life Technologies). A análise de expressão global dos miRNAs foi realizada utilizando a plataforma TaqMan Array Human MicroRNA Cards (TLDA) (card A, v3.0) (Life Technologies). Os dados de expressão foram extraídos através do programa Expression Suite Software v1.0.3. Análises através de softwares de bioinformática foram utilizadas para identificar genes-alvo de miRNAs, construindo redes de interação proteica. Resultados: Nosso estudo demonstrou um número maior de miRNAs desregulados (FC≥1,5 e p<0.05) na mucosa esofágica de pacientes com menos de 5 anos após a ingestão cáustica. 13 miRNAs (9 super- e 4 subexpressos) na mucosa esofágica de pacientes do Grupo A demonstraram-se significantemente desreguladas quando comparados a tecidos esofágicos normais. No Grupo B, 2 miRNAs apresentaram-se super- e 2 subexpressos. Destes, 2 miRNA (miR-374 and miR-574) estão relacionados de forma contundente ao câncer de esôfago segundo estudos recentes. Quando executada a rede de interação proteica, nossa análise evidenciou um número importante de interações entre miRNAs e genes- alvos entre as vias de sinalização molecular da neoplasia de esôfago, com genes fortemente associados à sua tumorigênese. Conclusões: A análise do perfil de expressão global de miRNAs nesta população pediátrica específica é inédita na literatura. Os miRNAs aqui identificados podem estar associados a biologia da transformação esofágica maligna da estenose cáustica para o cancer de esôfago, mesmo nos primeiros anos após a lesão, podendo servir no futuro como novos biomarcadores moleculares de progressão à malignidade. Estudos multicêntricos que reúnam maior tamanho amostral necessitam ser realizados para confirmar os resultados aqui obtidos e elucidar melhor o mecanismo subjacente e à correlação entre estenose cáustica e câncer de esôfago.
Background: 80% of the caustic ingestions occur in children. Esophageal stricture is a major chronic complication with great morbidity. Esophageal neoplasms may develop as a late complication of caustic injury with a mean time between caustic ingestion and cancer development of 15-30 years. Serial biopsies are recommended aiming early detection of premalignant changes. Thus a comprehensive knowledge of biological relation between caustic and esophageal cancer is of major importance to identify the biomarkers that could enable an early treatment. MicroRNAs (miRNAs) are small non-coding RNA molecules and regulate key cellular processes during tumorigenesis and have been demonstrated as an useful diagnostic, prognostic and predictive biomarkers. Global miRNA expression profiling analysis in this population, followed by the identification of miRNA target genes, may lead to the identification of the presence and magnitude of damage to genetic material in a sample of esophageal tissue obtained from patients with caustic stenosis. Objectives: We aimed to identify global microRNA (miRNA) expression changes in cells of the esophageal mucosa from children with caustic lesions compared to paired macroscopic and microscopically normal esophageal tissue. Patient and Methods: 27 formalin fixed, paraffin embedded (FFPE) esophageal samples from 15 patients were divided into two groups according to the time elapsed after the injury (Group A: less than 5 years, Group B: more than 5 years). Those tissues and their paired by gender, age and year of biopsy to normal (macro and microscopically) esophageal tissues . Group samples were needle microdissected and subjected to RNA isolation using the Recover AllTM Total Nucleic Acid Isolation Kit (Life Technologies). All samples were profiled using the TaqMan Array Human MicroRNA Cards (TLDA) (card A, v3.0) (Life Technologies). Data analysis was performed using ExpressionSuite Software v1.0.3. Computational bioinformatics analysis was performed to identify miRNA target genes, as well as to construct protein-protein interaction and miRNA-gene targets networks. Results: Our analysis showed a larger number of deregulated miRNAs (FC≥1,5 and p<0.05) in esophageal mucosa of patients with less than 5 years after caustic ingestion compared to those in with more than 5 years. 13 miRNAs (9 over- and 4 under-expressed) were significantly deregulated in Group A compared to paired normal esophageal tissue. In Group B, 2 miRNAs were under- and 2 were over-expressed. Of these, 2 microRNA (miR-374 and miR-574) are strongly related to esophageal cancer according to recent studies. When performed protein-protein interaction network of Group B), analysis showed a number of miRNA-target gene interactions within signal transduction and transcription factor networks in esophageal cancer, with genes strongly related to its tumorigenesis. Conclusions: There is no literature on the research of microRNAs in the population of patients who ingested caustics, especially children. miRNAs identified herein may be associated to the biology of the malignant transformation from caustic stenosis to esophageal cancer, even in early years after caustic lesion and may serve in the future as novel molecular markers for progression to malignancy. Further investigation is needed to elucidate the mechanism underlying the correlation and malignant transformation between caustic stenosis and cancer progression.
Background: 80% of the caustic ingestions occur in children. Esophageal stricture is a major chronic complication with great morbidity. Esophageal neoplasms may develop as a late complication of caustic injury with a mean time between caustic ingestion and cancer development of 15-30 years. Serial biopsies are recommended aiming early detection of premalignant changes. Thus a comprehensive knowledge of biological relation between caustic and esophageal cancer is of major importance to identify the biomarkers that could enable an early treatment. MicroRNAs (miRNAs) are small non-coding RNA molecules and regulate key cellular processes during tumorigenesis and have been demonstrated as an useful diagnostic, prognostic and predictive biomarkers. Global miRNA expression profiling analysis in this population, followed by the identification of miRNA target genes, may lead to the identification of the presence and magnitude of damage to genetic material in a sample of esophageal tissue obtained from patients with caustic stenosis. Objectives: We aimed to identify global microRNA (miRNA) expression changes in cells of the esophageal mucosa from children with caustic lesions compared to paired macroscopic and microscopically normal esophageal tissue. Patient and Methods: 27 formalin fixed, paraffin embedded (FFPE) esophageal samples from 15 patients were divided into two groups according to the time elapsed after the injury (Group A: less than 5 years, Group B: more than 5 years). Those tissues and their paired by gender, age and year of biopsy to normal (macro and microscopically) esophageal tissues . Group samples were needle microdissected and subjected to RNA isolation using the Recover AllTM Total Nucleic Acid Isolation Kit (Life Technologies). All samples were profiled using the TaqMan Array Human MicroRNA Cards (TLDA) (card A, v3.0) (Life Technologies). Data analysis was performed using ExpressionSuite Software v1.0.3. Computational bioinformatics analysis was performed to identify miRNA target genes, as well as to construct protein-protein interaction and miRNA-gene targets networks. Results: Our analysis showed a larger number of deregulated miRNAs (FC≥1,5 and p<0.05) in esophageal mucosa of patients with less than 5 years after caustic ingestion compared to those in with more than 5 years. 13 miRNAs (9 over- and 4 under-expressed) were significantly deregulated in Group A compared to paired normal esophageal tissue. In Group B, 2 miRNAs were under- and 2 were over-expressed. Of these, 2 microRNA (miR-374 and miR-574) are strongly related to esophageal cancer according to recent studies. When performed protein-protein interaction network of Group B), analysis showed a number of miRNA-target gene interactions within signal transduction and transcription factor networks in esophageal cancer, with genes strongly related to its tumorigenesis. Conclusions: There is no literature on the research of microRNAs in the population of patients who ingested caustics, especially children. miRNAs identified herein may be associated to the biology of the malignant transformation from caustic stenosis to esophageal cancer, even in early years after caustic lesion and may serve in the future as novel molecular markers for progression to malignancy. Further investigation is needed to elucidate the mechanism underlying the correlation and malignant transformation between caustic stenosis and cancer progression.
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Palavras-chave
estenose cáustica, miRNA, câncer de esôfago, cirurgia pediátrica, caustic stenosis, miRNA profiling, esophageal cancer, pediatric surgery