Comparing Canonical and Dicer Substrate Small Interfering RNA and Their Effects on the Silencing of Two Common Human Papillomavirus 16 E6 Variants
Master of Science
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High-risk human papillomaviruses (HPVs), such as HPV16, cause nearly all cases of cervical cancer as well as a number of head and neck cancers. Over expression of the E6 oncoprotein helps drive the malignant phenotype, therefore making it a promising therapeutic target. There are two common HPV16 E6 variants, Asian-American (AA) and European Prototype (EP), that differ by six single nucleotide polymorphisms (SNPs) resulting in AA having greater oncogenic potential than EP. Oncogene-specific down regulation mediated by RNA interference (RNAi) is an efficient approach that uses synthetic small interfering RNA (siRNA) to target and degrade E6 mRNA. Several E6 siRNAs were designed based on previously validated data, the Rosetta siRNA Design Algorithm, and Dicer substrate siRNA Design Algorithm. After transfection with the oligonucleotides in CaSki cells, Rosetta siRNA and Rosetta dicer substrate siRNA (DsiRNA) showed the highest E6 knockdown, having IC50 values of 7 ± 13 nM and 3 ± 2 nM, respectively. However, the differences between siRNA and DsiRNA were not statistically significant. Rosetta siRNA and Rosetta DsiRNA were then transfected into primary human foreskin keratinocytes (PHFKs) previously transduced with AA and EP, resulting in IC50 values of 3 ± 2 pM and 6 ± 3 pM respectively, for AA, and 72 ± 69 pM and 38 ± 2 pM respectively, for EP. E6 protein levels in both variants after treatment with either Rosetta siRNA or Rosetta DsiRNA were no longer detectable so to validate downstream process restoration once the E6 protein was knocked down, human telomerase reverse transcriptase (hTERT) mRNA and p53 protein levels were measured. Throughout this study, there was no significant difference between cells transfected with Rosetta DsiRNA and Rosetta siRNA when E6 mRNA, E6 protein, p53, and hTERT levels were measured by either RT-qPCR or western blot. After transfection with 6.75 nM of either Rosetta siRNA or Rosetta DsiRNA, p53 levels increased and hTERT levels decreased compared to negative controls. These findings suggest that Rosetta DsiRNA and Rosetta siRNA do not differ in downstream process restoration when targeting HPV16 E6 in vitro but may have an effect on duration of knockdown or stability of E6 mRNA in vivo.