RESUMEN
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.
Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorrectales/genética , Genes Relacionados con las Neoplasias , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-ets/fisiología , Adenocarcinoma/química , Adenocarcinoma/patología , Adenoma/química , Adenoma/patología , Anciano , Biomarcadores de Tumor/genética , Brasil , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Análisis de Matrices Tisulares , Transcriptoma , Ensayo de Tumor de Célula MadreRESUMEN
The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells.
