Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

Abstract

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.

Small interfering RNA s based on huntingtin trinucleotide repeats are highly toxic to cancer cells

“…When transfecting siCAG/CUG into various human (Fig B) and mouse (Fig C) cancer cell lines at 10 nM, all cancer cells stopped growing within hours of transfection and eventually most of the cells died with no outgrowth of recovering cells (Supplementary Movies EV1, EV2, EV3, EV4, EV5, EV6, EV7, EV8, EV9 and EV10; Appendix Fig S1A). All cancer cells transfected with siCAG/CUG showed morphological changes similar to the ones we observed in cells undergoing DISE (Appendix Fig S1B, ). We found that siCAG/CUG killed HCT116 cells even when transfected at 10 pM (Fig D).…”

“…Similar to cells undergoing DISE, when transfected with siCAG/CUG, ~1,800 critical survival genes but not ~400 nonsurvival control genes were significantly enriched in the downregulated genes in cells transfected with siCAG/CUG but not with siCGA/UCG in a gene set enrichment analysis (Fig D). In fact, we detected a ~12‐fold increased percentage of survival genes compared to the nonsurvival genes among the downregulated RNAs in the siCAG/CUG‐treated cells (Fig E)—a higher difference than seen in cells treated with DISE‐inducing sh‐ or siRNAs (data not shown).…”

“…P ‐values indicate the significance of enrichment. P ‐values were calculated using the Kolmogorov–Smirnov test. Venn diagrams showing the overlap between 1,185 survival genes (genes identified as critical survival genes in two genome‐wide lethality screens , blue) and genes significantly downregulated (red) in cells transfected with either siCAG/CUG (top) or siCGA/UCG (bottom) when compared to siNT. Metascape analysis of 4 RNA‐Seq datasets of cells with introduced siL3, two CD95L‐derived shRNAs (shL1 and shL3), a CD95‐derived shRNA (shR6) ORF, previously described , and the downregulated genes in cells transfected with siCAG/CUG. The boxed GO term clusters were highly enriched in all five datasets. Top, Venn diagram comparing the 3,466 genes downregulated in the siCAG/CUG‐treated HeyA8 cells (> 1.5‐fold adj.…”

Angiotensin II Silencing Alleviates Erectile Dysfunction Through Down-Regulating the Rhoa/Rho Kinase Signaling Pathway in Rats with Diabetes Mellitus

“…Six AngII shRNAs were designed according to the sequences of Genebank database and protocols from previous studies in order to select the two optimal sequences [15]. In brief, the expression vector was purchased from System Biosciences (Mountain View, CA, USA).…”

Long noncoding RNA complementarity and target transcripts abundance

“…The presence of Alu-repeats in human lincRNA-p21 [12] suggests that this change was due in part to hybridization of Alu-repeats in human lincRNA-p21 that target mRNAs through partial complementarity (Figure 2D). Although our microarray and RNA-seq analysis after siRNA transfection showed that the abundance of complementary target mRNAs changes, we cannot rule out an offtarget effect of siRNAs contributing to miRNA abundance, non-specifically [21]. It is also possible that the interaction between CCAACC DNA sequence and nuclear lincRNA-p21 could affect transcription of target mRNAs.…”