Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region
Fig 9
Requirement of pRNA transcription for gene expression.
(A) Specificity of ODN targeting:ODNs were checked for modulating bidirectional transcription in HeLa. HeLa cells were treated with respective ODNs followed by analysis of pRNA expression using directional qRT-PCR with P2 primer. SEN P2F: HeLa cells transfected with sense ODN and P2 forward primer (antisense pRNA specific) used in directional RT-PCR SEN P2R: HeLa cells transfected with sense ODN and P2 reverse primer (sense pRNA specific) used in directional RT-PCR, ANT P2F: HeLa cells transfected with antisense ODN and P2 forward primer (antisense pRNA specific) used in directional RT-PCR, ANT P2R: HeLa cells transfected with sense ODN and P2 reverse primer (sense pRNA specific) used in directional RT-PCR Control-ODN: Control ODN treated cells. Expression ratio was calculated with respect to 18S rRNA by REST software normalized to Control dsRNA treated samples. (B) Effect of sense or antisense pRNA knockdown on pRNA, E6 and E7 expression by ODN in HeLa. Control-ODN: Control ODN transfected cells, SEN-ODN: sense ODN transfected cells, ANT-ODN: antisense ODN transfected cells. (*) P < 0.01, (**) P < 0.05. C: Sense pRNA degradation mediated heterochromatization. Significant reduction in MNase digestion 72 hours after antisense ODN transfection while Control or sense ODN transfection had no effect in HeLa cells. Control-ODN: Control ODN transfected cells, SEN-ODN: cells transfected with sense ODN, ANT-ODN: cells transfected with antisense ODN, (*) P < 0.05.