Transcriptome-wide analysis of immune-responsive microRNAs against poly (I:C) challenge in Branchiostoma belcheri by deep sequencing and bioinformatics

Amphioxus is a key experimental animal for studying the evolution of vertebrate immune system. However, we still do not know about the roles of microRNAs (miRNAs) under viral stress in amphioxus. In this study, we sequenced six small RNA libraries (three biological replicates were included in the treatments challenged by the viral mimic, poly (I:C) (pIC) and control groups, respectively) from Branchiostoma belcheri. A total of 151 known miRNAs, 197 new miRNAs (named novel_mir, including nine conserved miRNAs) were identified by deep sequencing from the six libraries. We primarily focused on differentially expressed miRNAs (DEMs) after pIC challenge. Next, we screened a total of 77 DEMs, including 27 down- and 50 up-regulated DEMs in response to pIC challenge. Furthermore, we used real-time quantitative PCR (qRT-PCR) to verify the expression levels of 10 randomly selected DEMs. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses of these targets were performed using bioinformatics approach. MiRNA targets of DEMs are primarily involved in immune response, diseases, cancer and regulation process, and could be largely linked to 14 immune-related signaling pathways, including NF-kappa B, NOD-like receptor, RIG-I-like receptor and endocytosis. The present study for the first time explores key regulatory roles of miRNAs in the innate antiviral immune response in amphioxus, and will provide insight into the molecular basis of antiviral immunity and evolution of immune-related miRNAs.