Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer
Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer
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DOI：
10.1038/sj.onc.1206858
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发表时间：
2003-08-28
期刊：
影响因子：
8
通讯作者：
Liu, JS
中科院分区：
文献类型：
--
作者：
Yang, G;Thompson, JA;Liu, JS
为了研究H-ras在人卵巢癌发展中的作用，我们使用小抑制RNA （siRNA）沉默其在人卵巢癌细胞系中的表达，并评估其沉默对增殖、凋亡和肿瘤生长的影响。首先，我们开发了一种基于逆转录病毒的递送系统，允许长期稳定地表达所需的siRNA。在四种癌细胞系中，逆转录病毒介导的siRNA对绿色荧光蛋白（GFP）的表达降低了90%以上。然后，我们构建了三种逆转录病毒，表达针对H-rasV（12）突变的siRNA （H1/siRNA）或H-ras基因的两种野生型序列（H2/siRNA和H3/siRNA）中的任何一种，并用这些逆转录病毒感染T80H和SKOV-3细胞。在T80H细胞（一种基因转化的人卵巢表面上皮细胞，其致瘤性取决于H-rasV（12）的表达）中，H1/siRNA和H2/siRNA感染，而不是H3/siRNA感染，可降低T80H增殖，增加G(0)/G(1)阻滞和凋亡，在体外阻断转化，并抑制裸鼠肿瘤生长。在SKOV-3细胞（一种人类卵巢癌细胞系，含有高水平的野生型H-ras蛋白，但没有H-rasV（12）突变）中，引入H2/siRNA结构，而不是H1/siRNA或H3/siRNA，产生了类似的效果，表明siRNA对肿瘤生长的抑制是序列特异性的。我们得出结论，H-ras参与了人类卵巢癌肿瘤生长的维持，并且逆转录病毒介导的siRNA表达对H-Pas表达是解剖ras信号通路的有力工具，可能用于治疗卵巢癌。
To examine the role of H-ras in the development of human ovarian cancer, we used small inhibitory RNA (siRNA) to silence its expression in human ovarian cancer cell lines and assessed the effects of its silencing on proliferation, apoptosis, and tumorgrowth. First, we developed a retrovirus-based delivery system that allowed long-term stable expression of the desired siRNA. Retrovirus-mediated expression of siRNA against green fluorescence protein (GFP) reduced its expression more than 90% in four cancer cell lines. We then constructed three retroviruses that expressed siRNAs targeting the H-rasV(12) mutation (H1/siRNA) or either of two wild-type sequences of the H-ras gene (H2/siRNA and H3/siRNA) and used these retroviruses to infect T80H and SKOV-3 cells. In T80H cells (a genetically transformed human ovarian surface epithelial cell tine whose tumorigenicity depends on H-rasV(12) expression), infection with the H1/siRNA and H2/siRNA, but not with H3/siRNA, decreased T80H proliferation, increased G(0)/G(1) arrest and apoptosis, blocked transformation in vitro, and suppressed tumor growth in nude mice. In SKOV-3 cells (a human ovarian cancer cell line that contains high levels of wild-type H-ras protein but no H-rasV(12) mutation), introduction of the H2/siRNA construct, but not H1/siRNA or H3/siRNA, produced similar effects, demonstrating that the suppression of tumorgrowth by siRNA was sequence-specific. We conclude that H-ras is involved in maintenance of tumorgrowth of human ovarian cancer, and that retrovirus-mediated siRNA expression against H-Pas expression is a powerful toot to dissect ras-signaling pathways and may be used therapeutically against ovarian cancer.