Human papillomaviruses (HPV) type-16 and type-18 have been implicated in the causation and malignant progression of human cervical cancer. The consistent expression of two viral genes, E6 and E7, are required for the establishment and maintenance of a fully transformed phenotype. Recently, a powerful reverse genetic tool termed small silencing through RNA-mediated interference (RNAi) in mammalian cells. We together with others found that synthetic siRNAs targeted to various regions of HPV-16 E6 and E7 mRNA can specifically block the expression of respective viral oncogenes in HPV-positive tumor cell lines. However, the silencing of E6 and E7 expression by synthetic siRNA was transient. Silencing was sustained for only few days following a single dose of siRNA treatment. In our observation, suppression of E6 and E7 has caused a significant decrease in growth rate of HPV-positive tumor cells, but the continuous addition of siRNA in a daily basis was required. This transient reduction of gene expression severely restricts the therapeutic application of siRNAs. To overcome this limitation, we had generated two siRNA expression vectors to specifically target HPV-16 E6 and E7 mRNA. The endogenous siRNAs were found to inhibit the expression of cognate viral gene in tumor cell continuously. Moreover, the stable suppression of E6/E7 oncogene in HPV-positive TC-1 tumor cell successfully inhibited the cell growth in vitro and reduced the tumorigenicity of these cell in syngenic animal.
