miR-92b-5p negatively regulates IKK through targeting its ORF region in the innate immune responses of amphioxus (Branchiostoma belcheri)

Cephalochordate (Amphioxus), situated at a key phylogenetic position in the phylum Chordata, serves as a model organism for studying the origin and evolution of the vertebrate innate immune. In this study, five members of precursor miR-92 family (miR-92a-1, miR-92a-2, miR-92b, miR-92c and miR-92d) are identified in Branchiostoma belcheri, and their evolutionary conservation and potential molecular functions in innate immunity are analyzed. Among them, miR-92b-5p was validated in HEK293 cells to target the coding region but not classic 3′UTR of IKK (inhibitor of nuclear factor kappa-B kinase) mRNA, one integral component of MAPK and TLR4 immune signaling. Furthermore, the spatiotemporal expression patterns of miR-92b-5p and IKK were examined in different tissues or different time points (2 h, 4 h, 8 h, 12 h, 24 h and 48 h) post LPS stimulation at RNA and protein level in vivo. The seemingly inverse expression pattern between miR-92b-5p and IKK supports the involvement of miR-92b-5p in Branchiostoma belcheri innate immune response. In conclusion, our work not only illustrates the evolutionary pattern of Branchiostoma belcheri miR-92 family across chordates, but also reveals that miR-92b-5p could target IKK expression to regulate innate immune response.