title: Proteomic identification of LASP-1 down-regulation after RNAi urokinase silencing in human hepatocellular carcinoma cells.
abstract: In human hepatocellular carcinoma (HCC), the high expression of urokinase-type plasminogen activator (uPA) is an unfavorable prognostic factor and a therapeutic target. To identify the downstream effects of uPA silencing by RNA interference, we studied proteome modifications of uPA-inhibited SKHep1C3 cells, an HCC-derived cell line. The study with two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry showed Lim and SH3 protein 1 (LASP-1), cytokeratin 1 (CK-1), cytokeratin 10 (CK-10), and heterogeneous nuclear ribonucleoprotein H1 down-modulation after uPA inhibition. LASP-1, CK-1, and CK-10 are involved in cytoskeleton dynamics as heterogeneous nuclear ribonucleoprotein H1 takes part in the mRNA processing and stability. We first confirmed the proteomic data by Western blot and immunoflorescence and then explored the link between uPA and LASP-1. The ectopic expression of uPA and LASP-1 supported the proteomic results and showed that uPA up-regulation increased LASP-1 expression and that both were implicated in SKHep1C3 motility. siRNA LASP-1 inhibition showed that LASP-1 was involved in actin microfilaments organization of SKHep1C3 cells. The disruption of the actin microfilaments after LASP-1 depletion increased uPA secretion and SKHep1C3 motility. Our results would suggest the hypothesis that uPA and LASP-1 expression may be coordinated in HCC-derived cells. In summary, the proteomic identification of a set of uPA downstream proteins provides new insight into the function of uPA in HCC cells.
authors.affiliation: Department of Biomedical Sciences and Biotechnology, Division of Biology and Genetics, IDET Centre of Excellence, University of Brescia, Brescia, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Salvi
authors.forename: Italia
authors.initials: I
authors.lastname: Bongarzone
authors.forename: Francesca
authors.initials: F
authors.lastname: Miccichè
authors.forename: Bruna
authors.initials: B
authors.lastname: Arici
authors.forename: Sergio
authors.initials: S
authors.lastname: Barlati
authors.forename: Giuseppina
authors.initials: G
authors.lastname: De Petro
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D060588:LIM Domain Proteins; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D040901:Proteomics; D034622:RNA Interference; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C096353:LASP1 protein, human; D060588:LIM Domain Proteins; D014568:Urokinase-Type Plasminogen Activator