Silencing of HPV Viral Oncogenes E6 and E7 in Cervical Cancer
CD Spillane, L Kehoe, O Sheils, CM Martin, JJ O'Leary. Trinity College Dublin, Dublin, Ireland; The Coombe Women and Infant's Univercity Hospital, Dublin, Ireland
Background: The expression of the HPV oncogenes, E6 and E7, is necessary for the development of cervical cancer. After over 25 years of research many biological activities of E6 and E7 have been established, including their interaction with the major tumour suppressor proteins, p53 and pRb. However, a complete view of their oncogenic potential still eludes us.
Design: In this study, in order to identify downstream cellular targets of the viral oncogenes, short interfering RNA (siRNA) technology was employed to silence E6/E7 oncogenes in HPV16 transformed SiHa cells and subsequently the effect at the cellular transcriptome level was determined using microarray-based gene expression profiling. Five siRNA were designed towards the HPV16 E7 region. TaqMan® PCR specific for E7 and E6, western blotting of E6 and E7 targeted proteins, p53, p21 and Rb, and flow cytometry were applied to examine the effects of the E7 knockdown.
Results: With a concentration of just 10nM four out of five siRNA independently induced in excess of a 70% reduction in RNA levels of E7 and E6. There was also a significant increase in levels of p53, p21 and a decrease in hyperphosphorylated form of pRb indicating a reduction in E6 and E7 protein levels. The introduction of the E7 siRNA into the SiHa cells resulted in a stalling of the cell cycle in the G1 phase, phenotypic changes and an increase in cell autofluorescence, indicating the induction of senescence. Subsequently, the cellular transcriptome of two of the siRNA was assessed by genome-wide microarray analysis. In total 168 genes differentially expressed genes with a fold change values above 2 and FDR below 0.05 were commonly identified on both transcriptome profile. Of the 168 identified genes 158 were down-regulated and 10 up-regulated. A large fraction of these genes are involved in tumor-relevant processes, such as cell cycle regulation, DNA repair and spindle formation.
Conclusions: We describe an RNAi approach which leads to the suppression of HPV16 E6 and E7. This approach, which combines the use of siRNA-mediated gene silencing, microarray screening, and functional classification of differential genes, can be used in functional genomics study to elucidate the role of E6/E7 oncogene in the carcinogenesis of HPV16 and provide some possible targets for clinical treatment and drug development of cervical cancer.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Monday, March 22, 2010 1:00 PM
Poster Session II # 217, Monday Afternoon