RNA interference against urokinase in hepatocellular carcinoma xenografts in nude mice.
The serine protease urokinase-type plasminogen activator (u-PA) is overexpressed in hepatocellular carcinoma (HCC) and its expression level is inversely correlated with the patients' survival. The purpose of this study was to examine the effects of vector-based RNA interference (RNAi) of u-PA on the growth of human HCC xenografts in nude mice in order to investigate the role of u-PA in human HCC. Our results showed that the subcutaneous injection of small interfering RNAs (siRNA) u-PA SKHep1C3 stable transfected cells (pS siRNA u-PA) led to a growth delay in xenograft development, compared to those generated from empty vector; the molecular characterization of nodules (carried out by PCR, RT-PCR and immunohistochemical analysis) revealed the presence of plasmid DNA, the u-PA gene expression knockdown, at both mRNA and protein levels, giving evidence of a long-term and target-specific inhibition by vector-based RNAi 11 weeks after cell inoculation. We further studied the effects of u-PA down modulation on extracellular matrix (ECM) proteins evaluating the expression and organization of fibronectin (FN; one of the main ECM proteins). Immunohistochemical and immunofluorescence analysis of FN revealed FN fibrils in pS siRNA u-PA xenografts and in pS siRNA u-PA cells, thus identifying the FN fibril organization as a downstream effect of u-PA knockdown in this system.
Salvi
A
A
Biology and Genetics Division, Department of Biomedical Sciences and Biotechnology, IDET Centre of Excellence, University of Brescia, Brescia, Italy.
Arici
B
B
Alghisi
A
A
Barlati
S
S
De Petro
G
G
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D049109:Cell Proliferation; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays / Q000379:methods*
D005353:Fibronectins; D012333:RNA, Messenger; D014568:Urokinase-Type Plasminogen Activator