Abstract LB-243: microRNA mimics: siRNA lessons apply. miRNA mimics exhibit a significant off-target effect that is cell-line, length and dose dependent.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In light of the great success of siRNA reagents in modern research, similar reagents have also become increasingly popular to study miRNAs in cancer biology. To explore the endogenous function of a particular miRNA in fine-tuning target protein translation, synthetic mimics are transfected into host cells either to examine the effect of a candidate target 3′UTR on the expression of a reporter, or to identify new targets using microarray technology. Such studies typically employ a proprietary commercial universal negative control, whose suitability is taken for granted. Methods: We transfected commercial miRNA mimics into a large panel of breast cancer cell lines (BT549, Cama1, HCC1500, MCF7, MDAMB134, MDAMB231 MDAMB436, MFM223, SUM159, ZR75-1) to identify endogenous targets of miR-155 using microarray analysis. ‘Seed’ and passenger-strand mutants were integrated into the study as additional controls. Results: The resulting list of differentially-expressed genes often reflects a saturation of transcripts pertaining to a strong immune response to the transfected double-stranded RNA (dsRNA). Different cell lines exhibit variable sensitivity to dsRNA mimics, evident as the induction of the interferon pathway and apoptosis. Using custom variants of the mimics we show that this sequence-independent response is predominantly length-dependent and dose-dependent. Moreover, a substantial part of the remaining, seemingly sequence-specific response could be attributed to the passenger strand, assumed to be inert. Conclusions and future work: Commercial miRNA mimics may have significant off-target effects that require careful controls to account for the phenotypes under study. In particular, ‘seed’ mutants rather than universal negative controls are of essence. Additionally, alternative methods of transient miRNA over-expression are currently under investigation to validate targets identified to date. Taken together, these lessons have significant implications for all areas of cancer biology where synthetic mimic transfections are a means for investigating the functional role of miRNAs in the cell. Citation Format: Mae A. Goldgraben, Anna Git, Oscar Rueda, Carlos Caldas. microRNA mimics: siRNA lessons apply. miRNA mimics exhibit a significant off-target effect that is cell-line, length and dose dependent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-243. doi:10.1158/1538-7445.AM2013-LB-243