title: Off-target effects of plasmid-transcribed shRNAs on NFκB signaling pathway and cell survival of human melanoma cells.
abstract: Signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) are transcription factors involved in cell survival, inflammation and metastasis. Constitutively activated STAT3 is found in many cancers, including melanoma. To study the crosstalk between STAT3 and NFκB signaling and its role in regulation of cancer cell survival, we used RNA interference (RNAi) to down-regulate STAT3 expression in human melanoma cells. RNAi strategies including double-stranded RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA are widely used to knock down disease-causing genes in a targeted fashion. We found that shRNAs up-regulate non-specific NFκB activity, while siRNA directed against STAT3 specifically increase NFκB activity. The basal survival of melanoma cells is unaffected by STAT3 knockdown-likely due to activation of pro-survival NFκB signaling. Whereas, owing to off-target effects, plasmid-transcribed shRNA affects melanoma survival. Our data show that shRNA-mediated gene silencing induces non-specific or off-target effects that may influence cell functions.
authors.affiliation: Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093, Warsaw, Poland.
authors.forename: Kavita
authors.initials: K
authors.lastname: Ramji
authors.forename: Dorota Weronika
authors.initials: DW
authors.lastname: Kulesza
authors.forename: Salem
authors.initials: S
authors.lastname: Chouaib
authors.forename: Bozena
authors.initials: B
authors.lastname: Kaminska
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D016328:NF-kappa B; D034741:RNA, Small Interfering; D050796:STAT3 Transcription Factor