de Bock Charles E, Lin Zhen, Itoh Takashi, Morris David, Murrell George, Wang Yao
Orthopaedic Research Institute, Department of Medicine, St George Hospital, University of New South Wales, Sydney, NSW, Australia.
FEBS J. 2005 Jul;272(14):3572-82. doi: 10.1111/j.1742-4658.2005.04778.x.
The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G)-pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by approximately 93% and approximately 84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis.
尿激酶型纤溶酶原激活剂（uPA）受体（uPAR）参与信号转导和包括癌症转移、血管生成、细胞迁移及伤口愈合在内的生物学过程。它是其配体uPA的特异性细胞表面受体，uPA催化纤溶酶原形成纤溶酶，从而激活有助于细胞外基质降解的蛋白水解级联反应，这是癌症转移中的关键步骤。我们合成了三种不同的DNA酶（Dz372、Dz483和Dz720），它们在三个不同的嘌呤（A或G）-嘧啶（U或C）连接处靶向uPAR mRNA。其中两种DNA酶，Dz483和Dz720，在体外以低至1:0.2的摩尔比（uPAR与Dz）高效且特异地切割uPAR转录本。当用比uPAR转录本摩尔过量200倍的DNA酶进行2小时分析时，Dz720和Dz483能够分别在体外将uPAR转录本降低约93%和约84%。转染后，它们在人骨肉瘤细胞系Saos-2中也显示出切割uPAR mRNA的能力。DNA酶Dz720在转染后4小时内降低了uPAR mRNA，并使Saos-2细胞中的uPAR蛋白浓度降低了55%。通过体外基质胶试验评估，Dz720引起的uPAR mRNA和蛋白浓度降低显著抑制了Saos-2细胞的侵袭。DNA酶方法的应用为降低uPAR mRNA表达及抑制癌症侵袭和转移增添了一种新的潜在临床药物。
