Gondi Christopher S, Lakka Sajani S, Dinh Dzung H, Olivero William C, Gujrati Meena, Rao Jasti S
Program of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois, College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605, USA.
Neuron Glia Biol. 2004 May;1(2):165-76. doi: 10.1017/s1740925x04000237.
The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU(2)M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.
恶性胶质瘤向周围正常脑组织的弥漫性、广泛浸润被认为依赖于细胞外基质成分蛋白水解的改变。我们之前的结果清楚地表明，在肿瘤进展过程中，尿激酶型纤溶酶原激活物（uPA）、uPA受体（uPAR）和基质金属蛋白酶-9（MMP-9）的浓度显著增加，并且这些分子的反义稳定克隆可以抑制肿瘤生长。由于反义介导的基因沉默不能完全抑制靶mRNA的翻译，并且需要高浓度的反义分子才能实现基因沉默，因此在本研究中我们使用RNA干扰（RNAi）方法来沉默uPA、uPAR和MMP-9。我们研究了一种由巨细胞病毒（CMV）启动子驱动的DNA模板方法，以诱导发夹RNA（hpRNA）触发的RNAi，通过单一构建体抑制uPA、uPAR和MMP-9基因表达。在用表达针对uPAR、uPA和MMP-9的发夹小干扰RNA（siRNA）的质粒转染的细胞中，发现uPAR蛋白水平以及uPA和MMP-9的酶活性显著降低。通过免疫组织化学分析确定，与空载体和增强型病毒颗粒/慢病毒载体（EV/SV）转染的细胞相比，pU(2)M转染的SNB19细胞中uPA、uPAR和MMP-9的表达显著降低。此外，如免疫组织化学分析所示，针对这些分子的单一构建体的作用是特异性抑制它们各自的蛋白水平。在用表达针对uPA、uPAR和MMP-9的双链RNA（dsRNA）的质粒载体转染后，与空载体和EV/SV处理组相比，胶质瘤细胞侵袭受到抑制，这通过基质胶侵袭试验和球体侵袭试验得到证明。在体外（共培养）模型中，使用RNAi下调uPA、uPAR和MMP-9可抑制血管生成。向裸鼠预先建立的颅内肿瘤直接瘤内注射表达针对uPA、uPAR和MMP-9的hpRNA的质粒DNA可使肿瘤显著消退。此外，与亲本和EV/SV处理的SNB19细胞相比，用针对uPAR、uPA和MMP-9的RNAi处理的细胞显示磷酸化细胞外信号调节激酶（pERK）水平降低。我们的结果支持RNAi作为一种基因治疗方法在治疗胶质瘤方面的治疗潜力。
