MicroRNA mimics can distort physiological microRNA effects on immune checkpoints by triggering an antiviral interferon response

The microRNA-200 family has wide-ranging regulatory functions in cancer development and progression. Above all, it is strongly associated with the epithelial-to-mesenchymal transition (EMT), a process during which cells change their epithelial to a mesenchymal phenotype and acquire invasive characteristics. More recently, miR-200 family members have also been reported to impact the immune evasion of cancer cells by regulating the expression of immunoinhibitory immune checkpoints (ICs) like PD-L1. Therefore, we aimed to comprehensively characterize this miR-200 family as a regulatory interface between EMT and immune evasion mechanisms in biliary tract cancer. Initial correlation analyses and transient overexpression experiments using miRNA mimics suggested miR-200c-3p as a putative regulator of ICs including PD-L1, LGALS9, and IDO1. However, these effects could not be confirmed in stable miR-200c-3p overexpression cell lines, nor in cells transiently transfected with miR-200c-3p mimic from an independent manufacturer. By shifting our efforts towards dissecting the mechanisms leading to these disparate effects, we observed that the initially used miR-200c-3p mimic triggered a double-stranded (ds)RNA-dependent antiviral response. Besides upregulating the ICs, this had substantial cellular consequences including an induction of interferon type I and type III expression, increased levels of intracellular dsRNA sensors, and a significantly altered cellular growth and apoptotic activity.Our study highlights the capability of miRNA mimics to non-specifically induce a dsRNA-mediated antiviral interferon response. Consequently, phenotypic alterations crucially distort physiological miRNA functions and might result in a major misinterpretation of previous and future miRNA studies, especially in the context of IC regulation.