IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
mRNA Identification of a Novel Isoform (ISF) and ISF3 from ADAMTS13 in Different Types of Cells
Autor/es:
KEMPFER AC; DOS SANTOS C; PAIVA J; WOODS AI; LAZZARI MA; ALBERTO MF; SÁNCHEZ LUCEROS A
Lugar:
Berlin
Reunión:
Congreso; 26th Congress of ISTH & 63rd Annual Scientific and Standardization Committee (SSC) Meeting; 2017
Institución organizadora:
International Society on Thrombosis and Haemostasis
Resumen:
Background: ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF).Alternative splicing (AS) is a common feature of ADAMTS gene expression, in ADAMTS13 this process could product cleave the genomic sequence in different positions and causes several potential variants. Shonrom (2009) described mRNA of ISF1 in cancer lines and ISF1, ISF2 and ISF3 of Hep3B. We described previously (ISTH 2013) mRNA of ISF1 and ISF2 in Plts, HUVEC and ISF2 in two breast cancer lines (MDA-MB231, MCF7).Aims: Determine the presence of ISF3 in Plts, HUVEC and cancer cell lines, characterize and identify a potential ADAMTS13 ISF.Methods: Total RNA was isolated by TRIZOL method. The integrity was verified by 260/280 optical density ratio. mRNA was reverse transcribed using special primers. We designed primers to amplify the sequence of ADAMTS13 to differentiate Iso1 (421bp) and Iso3 (334bp). β-actin was used as control. Hep3B ISFs were used as control. The PCR products were analyzed on agarose gel containing SYBR Safe. IQTL software was used to quantitative analysis (Pixel intensity values=PIV).Results: All PCR performed, except the negative controls, were positive for β-actin and negative control did not present bands. A particular novel ISF (ED7-8) and ISF3 were observed in all cells studied: Plts, HUVEC, Hep3B, MDA-MB231 and MCF7 (Figure 1). The evaluation of PIV was made respect to ISF1 of each cell.The mean and standard deviation (SD) of three assays showed in Table I.The PIV results of ISF3 and ED7-8 respect to Hep3B were not statisticallysignificant for all the samples except for HUVEC. A large number of Plts PIV measurements would be necessary to decrease the high standard deviation obtained.Conclusions: The ISF 1, 3 and ED7-8 were identified by sequence analysis and quantified in relative form. Additional studies are also required to elucidate the relationship between the presence of the ADAMTS13 ISFs, translational regulation, protease activity and cells proliferation. (Reach the World Travel Grant)