Expression regulation analysis and functional characterization of HTRA1 in high risk HPV immortalized cells

Abstract

The Human Papillomavirus is the most prevalent virus among sexually transmitted infections and it is associated with various malignancies. The HPV genome has double stranded DNA and it is classified as low, intermediate or high oncogenic risk. The E6 and E7 proteins are the main responsible for cell homeostasis alteration, with consequent immortalization. Two of the different mechanisms used in cell transformation by the E6 protein are the transcricional control and interaction with PDZs domains of cellular proteins, triggering them to degradation. A protein that is associated with various pathological conditions and has PDZ domain is the protease HTRA1. This protein is poorly expressed in some cancers, suggesting a tumor suppressor role. The HTRA1 gene was selected for functional study because Dr. Luisa Lina Villa's group conducted a microarray where it was observed low expression of this gene in cell line HF698 immortalized by HPV16. Furthermore, we observed in our laboratory for quantitative PCR low expression of HTRA1 in cell line immortalized by HPV 16 and in samples of penile carcinoma infected with high-risk HPV. The aim of this study is to evaluate the effect of the HTRA1 overexpressed in cell line immortalized by HPV 16 (HF698), courtesy of Dr. Luisa Lina Villa, through analysis of protein expression of this gene after vector containing the HTRA1 ORF transfection, viability and cell proliferation assays, apoptosis assay and analysis of E6 and E7 expression from HPV 16 before and after vector overexpressing HTRA1 transfection. Furthermore, we will check if the E6 transcriptional silencing with siRNA increases HTRA1 expression. The analyzes to be performed are important for understanding the effect of HTRA1 overexpression in HPV 16 immortalized keratinocytes, seeking thereby to identify the cellular changes related with this gene expression. (AU)