Yang Gong, Thompson Jennifer Anne, Fang Bingliang, Liu Jinsong
Department of Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4095, USA.
Oncogene. 2003 Aug 28;22(36):5694-701. doi: 10.1038/sj.onc.1206858.
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 line 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-ras expression is a powerful tool to dissect ras-signaling pathways and may be used therapeutically against ovarian cancer.
为了研究H-ras在人类卵巢癌发生发展中的作用，我们使用小干扰RNA（siRNA）沉默其在人卵巢癌细胞系中的表达，并评估其沉默对细胞增殖、凋亡和肿瘤生长的影响。首先，我们构建了一种基于逆转录病毒的递送系统，该系统能够使所需的siRNA长期稳定表达。逆转录病毒介导的针对绿色荧光蛋白（GFP）的siRNA表达在四种癌细胞系中使GFP表达降低了90%以上。然后，我们构建了三种逆转录病毒，它们分别表达靶向H-rasV(12)突变的siRNA（H1/siRNA）或H-ras基因的两个野生型序列之一（H2/siRNA和H3/siRNA），并使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系，其致瘤性依赖于H-rasV(12)表达）中，用H1/siRNA和H2/siRNA感染，而不是用H3/siRNA感染，可降低T80H细胞的增殖，增加G(0)/G(1)期阻滞和凋亡，阻断体外转化，并抑制裸鼠体内肿瘤生长。在SKOV-3细胞（一种含有高水平野生型H-ras蛋白但无H-rasV(12)突变的人卵巢癌细胞系）中，导入H2/siRNA构建体，而不是H1/siRNA或H3/siRNA，产生了类似的效果，表明siRNA对肿瘤生长的抑制作用具有序列特异性。我们得出结论，H-ras参与人类卵巢癌肿瘤生长的维持，并且逆转录病毒介导的针对H-ras表达的siRNA表达是剖析ras信号通路的有力工具，可能用于卵巢癌的治疗。
