For co-transfections : 1 x 10^4 cells were seeded into 96-well plates, incubated overnight, and transfected with plasmids and RNAs using jetPRIME according to the manufacturer's instructions. For siRNA knock-down: 2 x 10^5 cells were seeded into 24-well plates, incubated overnight, and transfected with RNAs using INTERFERin according to the manufacturer's instructions.

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Previous reports have shown that some miRNAs are induced during virus infection and participate in the regulation of the innate antiviral response. However, whether the type I IFN response is regulated by miR-223 is still unknown. Here we reported that VSV infection induced significant upregulation of miR-223 in murine macrophages. We observed that miR-223 overexpression upregulated Type I IFN expression levels in VSV infected macrophages. And we demonstrated that miR-223 directly targets FOXO3 to regulate the Type I IFN production. Furthermore, Type I IFN, which is triggered by VSV infection, is responsible for the upregulation of miR-223, thus forming a positive regulatory loop for Type I IFN production. Our results uncovered a novel mechanism of miR-223-mediated regulation of Tydnape I IFN production in the antiviral innate immunity for the first time.