Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference

Abstract

Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.

The human papillomavirus E6 and E7 inducible oncogene, hWAPL, exhibits potential as a therapeutic target

“…In this study, we have demonstrated that an siRNA of hWAPL induces tumour regression in vivo. This anticancer effects by the hWAPL siRNA stands comparison with the effects of the E6 siRNA (Jiang and Milner, 2002;Butz et al, 2003;Yoshinouchi et al, 2003). Therefore, we expect that hWAPL is one of strong candidates as a target for uterine cervical cancer therapy.…”

“…For uterine cervical cancer, HPV E6 and/or E7 were shown to be a strong candidate as a target for gene-specific therapy (Jiang and Milner, 2002;Butz et al, 2003;Yoshinouchi et al, 2003). However, many subtypes of high-risk HPV have been detected in cervical cancers although HPV types 16 and 18 are great majority of cervical carcinomas (Nakagawa et al, 2000).…”

“…Therefore, the development of anticancer therapies that target HPV E6 and E7 and/or downstream targets of HPV E6 and E7 might be a specific and effective treatment for cervical cancer. In fact, RNA interference (RNAi) of E6 and/or E7 inhibits growth of HPV-positive cancer cells (Jiang and Milner, 2002;Butz et al, 2003;Yoshinouchi et al, 2003).Our previous study demonstrated that the novel human gene hWAPL showed the unscheduled high-level expression in cervical dysplasia and carcinoma (Oikawa et al, 2004). In addition, NIH3T3 cells overexpressing hWAPL developed into tumours upon injection into nude mice, suggesting that hWAPL plays a significant role in cervical carcinogenesis and tumour progression as an oncogene (Oikawa et al, 2004).…”

“…Therefore, the development of anticancer therapies that target HPV E6 and E7 and/or downstream targets of HPV E6 and E7 might be a specific and effective treatment for cervical cancer. In fact, RNA interference (RNAi) of E6 and/or E7 inhibits growth of HPV-positive cancer cells (Jiang and Milner, 2002;Butz et al, 2003;Yoshinouchi et al, 2003).…”

Human papillomavirus type 16 E6 protein interacts with cystic fibrosis transmembrane regulator-associated ligand and promotes E6-associated protein-mediated ubiquitination and proteasomal degradation

“…siRNA-mediated silencing of HPV16 E6 reduces endogenous CAL levels in Caski cells It has been reported that siRNA-mediated depletion of E6 or E6AP mRNA causes stabilization and nuclear accumulation of p53 (Jiang and Milner, 2002;Yoshinouchi et al, 2003). To show that CAL can be destabilized by E6 or E6AP under physiological conditions, we examined whether siRNAs against HPV16 E6 (siE6) or against E6AP (siE6AP) affected CAL in Caski cells, an HPV-positive cell line.…”