Evaluación de la transición epitelio-mesenquimal en la resistencia a 5-fluorouracilo en líneas celulares de cáncer colorrectal
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Date
2021
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Universidad Nacional de Trujillo
Abstract
El presente trabajo de investigación tuvo como objetivo caracterizar la Transición Epitelio Mesenquimal (TEM) en líneas celulares de cáncer colorrectal resistentes a 5-fluorouracilo (5-FU). Se utilizaron líneas celulares HCT116 y SW480 resistentes a 5-FU obtenidas en el trabajo previo de Tesis I; inmunofluorescencia por microscopía confocal, Western Blotting y Reacción en Cadena de la Polimerasa en tiempo real con Transcripción Reversa (RT-qPCR) para evaluar los niveles de expresión proteica y génica respectivamente, de marcadores epiteliales y mesenquimales; Wound Healing Assay en la determinación del potencial migratorio y análisis de correlación génica in silico utilizando herramientas bioinformáticas. Los análisis morfológicos por inmunofluorescencia mostraron que los grupos resistentes a 5-FU exhibieron disminución de E-cadherina, aumento de Snail, Vimentina en forma de haces y mayor β-catenina nuclear en concordancia con una menor expresión génica y proteica de marcadores epiteliales (E-Cadherina y Claudina-3), y aumento de marcadores mesenquimales, Vimentina y Slug. HCT116 resistente a 5-FU exhibió una elevada capacidad migratoria en comparación con el grupo parental, con el mayor porcentaje (48%) de reducción del área de la herida luego de 36 horas. Finalmente el análisis de correlación génica in silico muestra una posible relación entre los niveles de expresión del transportador ABCC1 y componentes de las vías de señalización Wnt/β-catenina y JAK/STAT. Se concluye que la resistencia adquirida al 5-FU puede inducir TEM (Transición Epitelio-Mesenquimal) en células de cáncer colorrectal, y ser responsable de las características de malignidad y progresión de la enfermedad.
ABSTRACT The present study aimed to characterize the Mesenchymal Epithelial Transition (TEM) in colorectal cancer cell lines resistant to 5-fluorouracil (5- FU). Cell lines HCT116 and SW480 resistant to 5-FU obtained in the previous work of Thesis I were used; immunofluorescence by confocal microscopy, Western Blotting and Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to evaluate the levels of protein and gene expression, respectively, of epithelial and mesenchymal markers; Wound Healing Assay in the determination of migratory potential and in silico gene correlation analysis using bioinformatics tools. Morphological analyzes by immunofluorescence showed that the resistant groups to 5-FU exhibited a decrease in E-cadherin, an increase in Snail, Vimentin in the form of bundles and a higher nuclear β-catenin in accordance with a lower gene and protein expression of epithelial markers (E -Cadherin and Claudin-3), and increased mesenchymal markers, Vimentin and Slug. HCT116 resistant to 5- FU exhibited a high migratory capacity compared to the parental group, with the highest percentage (48%) reduction in wound area after 36 hours. Finally, the in silico gene correlation analysis shows a possible relationship between the expression levels of the ABCC1 transporter and components of the Wnt / β-catenin and JAK / STAT signaling pathways. It is concluded that acquired resistance to 5-FU can induce TEM in colorectal cancer cells, and be responsible for characteristics of malignancy and disease progression.
ABSTRACT The present study aimed to characterize the Mesenchymal Epithelial Transition (TEM) in colorectal cancer cell lines resistant to 5-fluorouracil (5- FU). Cell lines HCT116 and SW480 resistant to 5-FU obtained in the previous work of Thesis I were used; immunofluorescence by confocal microscopy, Western Blotting and Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to evaluate the levels of protein and gene expression, respectively, of epithelial and mesenchymal markers; Wound Healing Assay in the determination of migratory potential and in silico gene correlation analysis using bioinformatics tools. Morphological analyzes by immunofluorescence showed that the resistant groups to 5-FU exhibited a decrease in E-cadherin, an increase in Snail, Vimentin in the form of bundles and a higher nuclear β-catenin in accordance with a lower gene and protein expression of epithelial markers (E -Cadherin and Claudin-3), and increased mesenchymal markers, Vimentin and Slug. HCT116 resistant to 5- FU exhibited a high migratory capacity compared to the parental group, with the highest percentage (48%) reduction in wound area after 36 hours. Finally, the in silico gene correlation analysis shows a possible relationship between the expression levels of the ABCC1 transporter and components of the Wnt / β-catenin and JAK / STAT signaling pathways. It is concluded that acquired resistance to 5-FU can induce TEM in colorectal cancer cells, and be responsible for characteristics of malignancy and disease progression.
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Keywords
Cáncer colorrectal. Transición epitelio-mesenquimal. 5- fluorouracilo. Quimioresistencia. ATP-binding casstte