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Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, IDET Centre of Excellence, University of Brescia, Brescia, Italy

Requests for reprints: Sergio Barlati, Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa n.11, 25123 Brescia, Italy. Phone: 0039-030-3717-241; Fax: 0039-030-3701-157. E-mail: barlati{at}med.unibs.it

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.

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Received 12/30/03; revised 2/27/04; accepted 4/ 2/04.