Anàlisi del paper de les glicosiltransferases (GT) en el fenotip tumoral de cèl•lules de càncer de pàncrees mitjançant el seu silenciament

Abstract

Nowadays cancer is a global problem being the second leading cause of deaths in the EEUU. Particularly, the pancreatic cancer (PDAC) causes the 7% of the deaths from cancer. The main problems of the PDAC are its delayed diagnostic and the low rate of survival in the advanced stages. It has been noticed that tumoral cells express abnormal glycosylation patterns in their surface. These changes include an overexpression of some glucidic antigens like Sialyl-Lewis. These antigens are composed by a Sialic acid molecule linked to the terminal chains of the Lewis sugars (antigens of the Lewis blood group). In fact, there are two types of Sialyl-Lewis antigens that have been observed to be overexpressed in tumoral cells: SLex and SLea. These antigens are synthesized by two α-2,3-sialyltransferases enzymes: ST3Gal III and ST3Gal IV. In this project, we want to analyse the role of the glycosyltransferases (ST3Gal III and ST3Gal IV) in the tumoral phenotype of pancreatic cancer cells. To achieve this objective, we want to perform a genomic knock-out of these enzymes using the CRISPR-Cas9 system and compare the phenotype of the control cells with the engineered cells. To verify the knock-out, we want to check the expression of ST3Gal III and ST3Gal IV at the protein level using a Western Blot analysis. To perform genomic editions, it is necessary to transfect BxPC-3 and Capan-1 cells with different vectors that contain the Cas9 nuclease and the sgRNA. For this reason, different liposomal reagents have been tested using different size vectors conjugated with GFP. The transfection protocol has been optimized to transfect our cells using a 5.4 kb vector equivalent to the vector with the sgRNA cloned in it. In contrast, using the 12.1 kb vector, equivalent to the vector with the Cas9 and the sgRNA cloned, we have achieved low transfection efficiencies. For these reasons, to perform the edition it has been established to transfect vectors with the sgRNA using the optimized protocol whereas Cas9 has to be introduced into our cells using another strategy. In addition, we have optimized the protocol to determine the ST3Gal III expression using the Western Blot technique. It has been established to use the TBST 5% of BSA blocking solution and low concentrations of secondary antibodies to get membranes showing the specific band of ST3Gal III