RNA Interference against Urokinase in Hepatocellular Carcinoma Xenografts in Nude Mice

Abstract

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 downmodulation 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.

Terminalia catappa attenuates urokinase-type plasminogen activator expression through Erk pathways in Hepatocellular carcinoma

“…Zheng et al showed that u-PA, u-PAR, and PAI-1 may be related to the invasion, metastasis, and prognosis of HCC [21]. Moreover, the u-PA targeting by molecular technologies (ex: antisense RNA or shRNA) [23,24], also in human HCC xenografts decreases the aggressive behavior of HCC cells inhibiting their migration and invasion capabilities [25]. In this study, we attempted to verify the u-PA molecular pathway of TCE in the TCE-mediated suppression of HCC metastasis.…”

MicroRNA‐23b mediates urokinase and c‐met downmodulation and a decreased migration of human hepatocellular carcinoma cells

“…uPA and c‐met are involved in human cancer, through the development and progression of several types of malignant tumor. An essential role of uPA and c‐met in the migration and proliferation of human hepatocellular carcinoma (HCC) cells has been assessed by functional studies carried out using RNA interference technology [18,19]. The evidence suggests that the two systems, uPA–uPA receptor and HGF–c‐met, might cooperate in the acquisition of malignant phenotypes of cancer cells, but little is known about the regulatory mechanisms for expression of these genes [20,21].…”

Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma

“…Other studies aimed at silencing the serine protease urokinase-type plasminogen activator (u-PA) demonstrated similar proof of principle efficacy [99,100] . Signalling through u-PA and its rece ptor (uPAR) have been implicated in cell invasion, survival, and metastasis of a variety of cancers [101,102] .…”

“…Moreover, silencing of u-PA resulted in attenuation of malignancyassociated cellular properties, such as migration, invasion and proliferation. In a follow up study, the effects of stable inhibition of u-PA on xenografted tumor cells were assessed [99] . Cells that stably produce silencing sequences targeted to u-PA were injected subcutaneously into nude mice.…”