Inhibition of PAI-1 expression in breast cancer carcinoma cells by siRNA at nanomolar range

Plasminogen activator inhibitor type I (PAI-I) plays a central role in metastatic behavior by increasing cells' migratory capacities as shown in several tumoral cell lines. Moreover, in vivo high expression of this factor helps tumoral growth, both by its role in extracellular matrix remodeling and by favoring angiogenesis. High levels of PAI-I are correlated with bad prognosis in several cancers, particularly in breast cancer. The effect of PAI-1 upon angiogenesis is also involved in atherosclerosis, in which high levels of PAI-I expression are observed. Breast carcinoma MDA MB 231 cells are known for both having important metastatic capacities and expressing high levels of PAI-1. We have demonstrated in these cells that the transfection of PAI- I specific small interfering RNAs (siRNA) specifically inhibited the expression of this factor by 91%. We evaluated siRNA activity by determining PAI- I mRNA level, as well as intracellular and extracellular PAI- I protein by using RT Q-PCR, Western blot and ELISA analyses, respectively. Data confirmed inhibition at mRNA levels (primary aim of interference), intracellular protein, and secreted PAI-I, the latter being operative successfully in the cell microenvironment. The lipidic vector Delivery Liposomes System (DLS) used was adapted to siRNA delivery as observed by particle size distribution analysis, confocal microscopy and transfection into MDA MB 23 1, in the presence of serum. SiRNA activity was clearly detected at concentrations as low as 10 nM. Moreover, the low cytotoxicity of this vector makes it a good candidate for future in vivo siRNA delivery. (c) 2007 Elsevier Masson SAS. All rights reserved.