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.

Integration of leave-one-out method and real-time live cell reporter array system to assess the toxicity of mixtures.

The ever-increasing number of chemicals and complex mixtures demands a time-saving and cost-effective platform for environmental risk assessment. However, there is limit promising tool for evaluating the contribution of each component to the total toxicity effects of the mixture. Here, four widely distributed environmental pollutants with different mode-of-actions, i.e., cadmium chloride (Cd), nitrofurazone (NFZ), triclosan (TCS), and tris(2-chloroethyl) phosphate (TCEP), were selected as components of artificial mixture. Integration of leave-one-out method and high-dimensional live cell array system was used to explore relative contribution of each component from the mixture. A quaternary mixture (All_4_chems) and four ternary mixtures (Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP) were investigated by Escherichia coli (E. coli) live cell array system with 90 environmental stress genes modified by green fluorescent protein (GFP) expressing reporter vectors. E. coli cytotoxicity tests demonstrated that TCS has antagonism effect with other three chemicals (Cd, NFZ and TCEP), while it was additive effect in other three binary combinations. A total of 26, 23, 13, 31 and 23 genes were significantly altered with fold-change greater than 2 over the 4 h exposure by All_4_chems, Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP, respectively. Clustering analysis based on time-series gene expression patterns and transcriptional effect level index (TELI) showed that Leave_TCEP has similar profiles with All_4_chems, demonstrating TCEP has the least contribution among four components to the quaternary mixture. Leave_NFZ has the least number of significantly altered genes, implying NFZ has the largest toxicity effect contribution to the quaternary mixture. The relative contribution in different pathways indicated that Cd has the most contribution to the mixture in redox stress, while TCS has the least contribution in DNA stress pathway. Collectively, our results demonstrated the utility of high-dimensional toxicogenomics data and leave-one-out method in prioritizing the relative contribution of each component in mixture.

miR-2013 negatively regulates phylogenetically conserved PIP5K involved in TLR4 mediated immune responses of amphioxus (Branchiostoma belcheri Tsingtaunese).

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a catalytic kinase that performs multiple functions in organisms. Recent studies have shown that PIP5Kα in mammals can directly participate in the TLR-mediated innate immune regulation by controlling the production of PIP2. However, the PIP5K homologous gene has not been identified in Cephalochordata to date. In this study, we firstly identify and characterize a new PIP5K family member (designed as AmphiPIP5K) from Cephalochordata amphioxus (Branchiostoma belcheri tsingtaunese), particularly AmphiPIP5K is orthologous with vertebrate PIP5Kα and paralogous with PIP5Kß and PIP5Kγ. Secondly, we find that the AmphiPIP5K is involved in amphioxus innate immune response to LPS stimulation. Thirdly, our results demonstrate that miR-2013 can inhibit AmphiPIP5K expression by binding to the CDS and 3' UTR regions of AmphiPIP5K. Collectively, our work not only demonstrates the evolutionary pattern of amphioxus PIP5K, but also reveals that miR-2013 negatively regulate PIP5K expression to involve in amphioxus innate immune response.