Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis
Heparanase gene silencing, tumor invasiveness, angiogenesis, and metastasis
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DOI：
10.1093/jnci/djh230
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发表时间：
2004-08-18
影响因子：
10.3
通讯作者：
Vlodavsky, I
中科院分区：
文献类型：
--
作者：
Edovitsky, E;Elkin, M;Vlodavsky, I
背景资料：乙酰肝素酶是一种内切糖苷酶，其降解硫酸乙酰肝素，硫酸乙酰肝素是细胞外基质和基底膜的主要多糖成分。乙酰肝素酶基因的表达与多种恶性肿瘤和细胞系的侵袭性、血管生成和转移潜能相关。我们使用基因沉默策略来评估乙酰肝素酶在恶性肿瘤中的作用，并探索其特异性靶向治疗的潜力。研究方法：我们设计了质粒载体来表达锤头状核酶或针对人或小鼠乙酰肝素酶mRNA的小干扰RNA（siRNA）。用抗乙酰肝素酶核酶或siRNA转染人乳腺癌（MDA-MB-435）和小鼠淋巴瘤（Eb）和黑素瘤（B16-BL 6）肿瘤细胞系，所述肿瘤细胞系具有天然高水平的内源性乙酰肝素酶或已被遗传工程化以过表达乙酰肝素酶。半定量逆转录-聚合酶链反应（RT-PCR）和酶活性的测量被用来确认乙酰肝素酶基因表达的有效沉默。测试用抗乙酰肝素酶核酶和siRNA载体转染的细胞的体外侵袭性和在实验性和自发转移的动物模型中的转移性播散。结果如下：与转染对照构建体的细胞相比，转染抗乙酰肝素酶核酶或siRNA载体的细胞在体外显著降低了侵袭和粘附，无论细胞类型如何，并且表达较少的乙酰肝素酶。在体内，由用抗乙酰肝素酶核酶和siRNA载体转染的细胞产生的肿瘤比由用对照载体转染的细胞产生的肿瘤血管化更少且转移性更低。注射Nvith转染抗乙酰肝素酶核酶和siRNA载体的细胞的小鼠比注射对照细胞的小鼠寿命更长。结论：抗乙酰肝素酶核酶或siRNA介导的基因沉默载体与细胞中乙酰肝素酶水平降低和致瘤性改变的相关性表明乙酰肝素酶在癌症进展中很重要。乙酰肝素酶基因沉默作为抗癌药物开发的靶点具有潜在的用途。
Background: Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. Expression of the heparanase gene is associated with the invasive, angiogenic, and metastatic potential of diverse malignant tumors and cell lines. We used gene-silencing strategies to evaluate the role of heparanase in malignancy and to explore the therapeutic potential of its specific targeting. Methods: We designed plasmid vectors to express hammerhead ribozymes or small interfering RNAs (siRNAs) directed against the human or mouse heparanase mRNAs. Human breast carcinoma (MDA-MB-435) and mouse lymphoma (Eb) and melanoma (B16-BL6) tumor cell lines, which have naturally high levels of endogenous heparanase or have been genetically engineered to overexpress heparanase, were transfected with anti-heparanase ribozyme or siRNA. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and measurements of enzymatic activity were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with the anti-heparanase ribozyme and siRNA vectors were tested for invasiveness in vitro and metastatic dissemination in animal models of experimental and spontaneous metastasis. Results: Compared with cells transfected with control constructs, cells transfected with the anti-heparanase ribozyme or siRNA vectors had profoundly reduced invasion and adhesion in vitro, regardless of cell type, and expressed less heparanase. In vivo, tumors produced by cells transfected with the anti-heparanase ribozyme and siRNA vectors were less vascularized and less metastatic than tumors produced by cells transfected with the control vectors. Mice injected with cells transfected Nvith the anti-heparanase ribozyme and siRNA vectors lived longer than mice injected with control cells. Conclusions: The association of reduced levels of heparanase and altered tumorigenic properties in cells with anti-heparanase ribozyme- or siRNA-mediated gene-silencing vectors suggests that heparanase is important in cancer progression. Heparanase gene silencing has potential use as a target for anticancer drug development.