Salvi Alessandro, Arici Bruna, De Petro Giuseppina, Barlati Sergio
Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa n.11, 25123 Brescia, Italy.
Mol Cancer Ther. 2004 Jun;3(6):671-8.
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.
丝氨酸蛋白酶尿激酶型纤溶酶原激活物（u-PA）参与多种生理和病理过程；特别是，u-PA mRNA在人类肝细胞癌（HCC）活检组织中上调，其表达水平与患者生存率呈负相关。为了确定u-PA在HCC侵袭特性中的作用，我们通过RNA干扰（RNAi）技术在u-PA表达水平高的HCC衍生细胞系中成功下调了u-PA。RNAi是一个多步骤过程，涉及产生导致靶基因特异性抑制的小干扰RNA（siRNA）。将SKHep1C3细胞用编码由两个反向的相同19个核苷酸序列基序组成的RNA的U6启动子质粒转染，这两个基序由一个9碱基对的间隔区隔开，形成能够介导靶u-PA抑制的发夹双链RNA。稳定转染细胞显示u-PA蛋白水平持续降低。在生物学试验中，转染siRNA u-PA的细胞迁移、侵袭和增殖减少。总之，通过RNAi技术下调u-PA可降低HCC细胞的侵袭能力，表明siRNA u-PA的稳定表达可能是HCC基因治疗的一种实验方法。
