No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA.
Gene silencing by RNAi and siRNAs has become a well-used tool for researchers. Because of its relatively small size, siRNA was originally thought to avoid activation of anti-viral responses. Recent reports demonstrating so-called "off-target effects" are therefore alarming. One issue raised is that siRNA induces interferon-regulated genes at the transcriptional level. We characterize the anti-viral responses of synthetic siRNA and in vitro-transcribed siRNA by measuring the mRNA levels of IFN-beta and OAS2 in HeLa cells. Transfections with both traditional and LNA-modified synthetic siRNA cause no anti-viral responses, whereas transfection with either long dsRNA or in vitro-transcribed siRNA leads to greater than 1000-fold induction of these genes. The lack of response was also demonstrated at the level of phosphorylated eIF2alpha, and measuring of IFN-beta by ELISA in cell culture media from the human cell line MCF-7. Altogether, transfection with synthetic siRNA does not induce anti-viral responses in these two cell lines. Our results reinforce the role of siRNA as an effective tool for reverse genetics and strengthen siLNA as a tool for future therapeutic applications.
Department of Cell and Microbiology, Programme for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, S-171 77 Stockholm, Sweden. cecilia.dahlgren@ki.se
Cecilia
C
Dahlgren
Claes
C
Wahlestedt
Håkan
H
Thonberg
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells / Q000502:physiology*; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis; D009711:Nucleotides / Q000235:genetics*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D012266:Ribose / Q000031:analogs & derivatives* / Q000235:genetics; D014162:Transfection / Q000379:methods*
C434798:2'-O-4'-C-methylene-ribofuranosyl nucleotide; D009711:Nucleotides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; C477371:locked nucleic acid; D012266:Ribose; D016899:Interferon-beta; D015088:2',5'-Oligoadenylate Synthetase