Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer

Abstract

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.