Study of the promoter regulation, the target transcriptome and the physiological and pathological function of the HABP4 protein, involved in colon cancer.

Abstract

HABP4 is an unstructured protein that shows high plasticity and interaction with several targets, genes and proteins. Recent studies suggest that this protein is related to tumorigenesis, since the increase or decrease of its expression influences cell proliferation, cell cycle control and apoptosis. The Habp4 gene is located on chromosome 9, linked to four other genes, forming a haplotype for familial colon cancer. Colorectal cancer, in its turn, is the third most incident and the second most mortal. Some mutations in the Habp4 gene are related to this cancer. Considering the above, the present work aims to study what regulates the expression of HABP4, the targets regulated by it and the HABP4-protein interaction in human colorectal carcinoma cells (HCT116), under different stressful conditions. In addition, it is intended to evaluate variants of HABP4 mutants and their implications on HCT116 cells. The sequences of the Habp4 promoter and of the transcription factor binding sites will be investigated by in silico and in vitro analysis. The transcription factors that interact with the promoter region will be identified by reporter gene expression. Therefore, HCT116 cells will be transfected with a vector containing the Habp4 promoter and the luc2 reporter gene, and then subjected to stressful conditions. The influence of HABP4 on other genes regulation will be investigated by analyzing the general transcriptome of wild or knockout HCT116 cells, submitted or not to stressful conditions. For this, the RNA will be extracted and than sequenced. The HABP4-protein interaction in HCT116 cells will be performed using the BioID technique, followed by analysis in mass spectrometry. Finally, the influence of HABP4 on cell viability and on cell cycle in HCT116 will be evaluated from site-directed mutations in the Habp4 gene, and wild HCT116 cells will be compared with the different Habp4 mutant cells. Considering the relationship of this gene/protein with colorectal cancer and the relevance of this disease to society, the present study becames pertinent, allowing a better understanding of the assossiation bwetween the Habp4 gene and familial colon cancer; and promising for new research that considers this gene as a therapeutic target for this type of cancer.