Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells

Abstract

RNA interference-mediated gene silencing has the potential to block gene expression. A synthetic double-stranded small interfering RNA (siRNA) based on a sequence motif of 21 nucleotides from human papillomavirus 16 (HPV16) E6E7 bicistronic RNA was found to be a potent siRNA that suppresses expression of both the E6 and E7 oncogenes in HPV16þ CaSki and SiHa cells. When stably expressed as a short hairpin RNA in these cells, however, siRNA silencing of E6 and E7 expression was efficient only at early cell passages, but became inefficient with increased cell passages despite the continued expression of the siRNA at the same level. The loss of the siRNA function was duplicable in stable p53 siRNA cells, but not in stable lamin A/C siRNA cells, suggesting that it is gene selective. The cells resistant to siRNA function retained normal siRNA processing, duplex unwinding and degradation of the unwound sense strand and RNA-induced silencing complex formation, suggesting that loss of the siRNA function occurred at a later step. Surprisingly, the siRNA-resistant cells were found to express notably a cytoplasmic protein of B50 kDa that specifically and characteristically interacted with the unwound, antisense strand E7 siRNA. Altogether, our data indicate that a potent siRNA targeting to an essential or regulatory gene might induce a cell to develop siRNA-suppressive function.

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

“…20 and our data above demonstrated that shRNA delivered by lentivirus was also short-lived. One strategy to overcome this issue is the multi-copy or multi-target approach, which allows additive or even synergistic effects to induce rapid cell death before resistance occurs.…”

“…18,19 In addition to the above issue, cervical cancer cell lines CaSki and SiHa have been reported to be able to develop resistance to RNAi through the expression of a previously unknown 50 kDa cytoplasmic protein that specifically interacted with the unwound antisense strand of E7 siRNA from the RNA-induced silencing complex. 20 However, this specific interaction was not observed in lamina A/C gene. Therefore the authors considered this was a cellular protection reaction to E7 as it is essential for the cell to survive.…”

“…19 Another potential explanation is that the transduced HeLa cells have developed resistance to RNAi, as previously described in cervical cancer cells. 20 Whatever the reason, the data indicates that in cervical cancer, lentiviral vector can only deliver a short-lived RNAi therapy of about 2 weeks.…”

“…Therefore the authors considered this was a cellular protection reaction to E7 as it is essential for the cell to survive. 20 The resistance was not only seen in siRNA treatment but was also seen in shRNA. 21 Such resistance in cervical cancer cells could be an obstacle for the development of RNAi-based treatment.…”

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor

“…Total RNAs were purified at 24 h after transfection. Small RNAs were then gel purified and cloned by using the adapter ligation method (26). Approximately 100 g of total RNA from transfected or infected cells was separated on a 12% denaturing PAGE gel before transfer to a membrane and hybridization with 32 P-labeled oligonucleotide probes as described previously (26).…”