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Authors
Abstract(s)
O cancro do pulmão é um dos mais diagnosticados e mortais em todo o mundo. Os protooncogenes desempenham funções importantes tais como reguladores do crescimento
celular normal, a divisão e a apoptose. No entanto os proto-oncogenes podem dar origem a
oncogenes e levar ao crescimento celular descontrolado, o que pode resultar no
desenvolvimento de cancro. Oncogenes têm um papel crucial no desenvolvimento do cancro.
B-MYB é um proto-oncogene que codifica um fator de transcrição crucial na proliferação e
diferenciação celular. Em células do cancro do pulmão este gene encontra-se com níveis de
expressão elevados. De forma a controlar a expressão de oncogenes tem surgido interesse
em estudar estruturas não-canónicas de ácidos nucleicos. A estrutura i-motif (iM) tem
interesse como possível alvo terapêutico, uma vez, que há sugestões que indicam que estas
estruturas estão envolvidas em processos biológicos como a transcrição.
Este trabalho teve como objetivo identificar e caracterizar a estrutura iM no gene B-MYB
assim como estudar a interação/estabilização na presença de ligandos.
De maneira a alcançar estes objetivos, foi primeiramente realizada uma análise
bioinformática do gene B-MYB onde foram identificadas regiões promissoras de estruturas
de iM. Após a análise bioinformática foram realizados estudos de caracterização biofísica
tais como dicroísmo circular (CD), UV, ressonância magnética nuclear e PAGE.
Posteriormente foi determinada a estabilização das sequências na presença de vários
ligandos (acridinas, porfirinas, fenantrolinas) por FRET-melting e CD e afinidade através
da técnica de ressonância plasmónica de superfície (SPR) baseada num biossensor ótico e
espetroscopia de fluorescência. Por fim foi estudada a interação dos ligandos selecionados
(TMPyP4 e 360A) na linha celular A549.
Os resultados da análise bioinformática demonstraram 6 regiões promissoras para a
formação da estrutura iM. Através da caracterização biofísica foi demonstrado que 5 formam
a estrutura iM. O ensaio da interação de ligandos na linha celular A549 mostrou que o
ligando TMPyP4 induz a formação da estrutura i-motif nas células. O ligando 360A não
apresentou grande diferença em relação ao controlo, concluindo assim que este ligando não
induz a formação do iM nesta linha celular.
Lung cancer is one of the most diagnosed and deadliest cancers worldwide. Proto-oncogenes have important functions, including regulating normal cell growth, division and apoptosis. However, proto-oncogenes have the potential to become oncogenes, which can cause uncontrollable cell growth, ultimately leading to the development of cancer. The presence of oncogenes plays a crucial role in the pathogenesis of cancer. B-MYB is a proto-oncogene, displaying high expression levels in lung cancer, that encodes a transcription factor crucial in cell proliferation and differentiation. There is increasing interest in investigating non-canonical nucleic acid structures to regulate oncogene expression, with the i-motif (iM) structure of particular interest as a possible therapeutic target. This arises from suggestions that these structures play a role in biological processes such as transcription. This work aimed to identify and characterize the iM structure of the B-MYB gene, as well as explore its interaction and stabilization in the presence of ligands. To achieve these objectives, a bioinformatic analysis of the B-MYB gene was first carried out, in which was identified promising regions of iM structures. Subsequently, biophysical characterization studies were conducted, including circular dichroism, nuclear magnetic resonance, PAGE and UV. The sequences’ stability in the presence of different ligands - acridines, porphyrins, phenanthrolines- was assessed using FRET and CD, and SPR technique to determine their affinity. Thereafter, the interaction of the selected ligands (TMPyP4 and 360A) was studied in the A549 cell line. The bioinformatics analysis revealed 6 promising regions for iM structure formation, with biophysical characterization confirming that 5 of those regions form the iM structure. The ligand interaction assay on the A549 cell line showed that the TMPyP4 successfully induces i-motif structure formation in the cells. However, no significant difference was observed when the 360A ligand was compared to the control, concluding that this ligand does not induce iM formation in this cell line.
Lung cancer is one of the most diagnosed and deadliest cancers worldwide. Proto-oncogenes have important functions, including regulating normal cell growth, division and apoptosis. However, proto-oncogenes have the potential to become oncogenes, which can cause uncontrollable cell growth, ultimately leading to the development of cancer. The presence of oncogenes plays a crucial role in the pathogenesis of cancer. B-MYB is a proto-oncogene, displaying high expression levels in lung cancer, that encodes a transcription factor crucial in cell proliferation and differentiation. There is increasing interest in investigating non-canonical nucleic acid structures to regulate oncogene expression, with the i-motif (iM) structure of particular interest as a possible therapeutic target. This arises from suggestions that these structures play a role in biological processes such as transcription. This work aimed to identify and characterize the iM structure of the B-MYB gene, as well as explore its interaction and stabilization in the presence of ligands. To achieve these objectives, a bioinformatic analysis of the B-MYB gene was first carried out, in which was identified promising regions of iM structures. Subsequently, biophysical characterization studies were conducted, including circular dichroism, nuclear magnetic resonance, PAGE and UV. The sequences’ stability in the presence of different ligands - acridines, porphyrins, phenanthrolines- was assessed using FRET and CD, and SPR technique to determine their affinity. Thereafter, the interaction of the selected ligands (TMPyP4 and 360A) was studied in the A549 cell line. The bioinformatics analysis revealed 6 promising regions for iM structure formation, with biophysical characterization confirming that 5 of those regions form the iM structure. The ligand interaction assay on the A549 cell line showed that the TMPyP4 successfully induces i-motif structure formation in the cells. However, no significant difference was observed when the 360A ligand was compared to the control, concluding that this ligand does not induce iM formation in this cell line.
Description
Keywords
B-Myb Biofísica Cancro do Pulmão I-Motif Ligandos