Small interfering RNA urokinase silencing inhibits invasion and migration of human hepatocellular carcinoma cells.
The serine protease urokinase-type plasminogen activator (u-PA) is involved in a variety of physiologic and pathological processes; in particular, u-PA mRNA is up-regulated in human hepatocellular carcinoma (HCC) biopsies and its level of expression is inversely correlated with patients' survival. To determine the role of u-PA in the invasiveness properties of HCC, we successfully down-regulated u-PA by RNA interference (RNAi) technology, in an HCC-derived cell line at high level of u-PA expression. RNAi is a multistep process involving generation of small interfering RNAs (siRNA) that cause specific inhibition of the target gene. SKHep1C3 cells were transfected with a U6 promoter plasmid coding for an RNA composed of two identical 19-nucleotide sequence motifs in an inverted orientation, separated by a 9-bp spacer to form a hairpin dsRNA capable of mediating target u-PA inhibition. Stable transfectant cells showed a consistently decreased level of u-PA protein. In biological assays, siRNA u-PA-transfected cells showed a reduction of migration, invasion, and proliferation. In conclusion, u-PA down-regulation by RNAi technology decreases the invasive capability of HCC cells, demonstrating that stable expression of siRNA u-PA could potentially be an experimental approach for HCC gene therapy.
Salvi
Alessandro
A
Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa n.11, 25123 Brescia, Italy.
Arici
Bruna
B
De Petro
Giuseppina
G
Barlati
Sergio
S
D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D006801:Humans; D009361:Neoplasm Invasiveness / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism
C524898:PLAUR protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D014568:Urokinase-Type Plasminogen Activator