| TLR(Toll-like receptor)signaling pathway plays an important role in the innate immunity of animal.In recent years,amphioxus has gradually become a model organism to investigate animal immune system due to its intricate innate immune system and the prototype of specific adaptive immunity.Micro RNA(mi RNA)is a class of non-coding small-molecule RNA that has regulatory functions in animals,and the mi R-92 family are reported to be involved in the regulation of immunity.The field of amphioxus(Branchiostoma belcheri)mi RNA research mainly focuses on characterization of immune-related mi RNA through high-throughput RNA-sequencing method with limited functional studies.Considering the special evolutionary status of amphioxus,this study focuses on the amphioxus mi R-92 family participating in the innate immune regulation of amphioxus TLR pathway,and further analyzes the evolution of mi R-92 family.We obtained the following results:1.This study analyzed the phylogeny of the mi R-92 family.Taking the mi R-92 family of amphioxus as an example,the stability of the stem-loop structure of the precursor mi RNA that may be encoded is predicted.Alignment of mi R-92 family sequences of different species found that this mi RNA family exists in almost all vertebrate and invertebrate species.The sea urchin has the largest mi R-92 b family members;the conservation of amphioxus bbe-mi R-92 b relative to mi R-92 b of other specie s is more conserved than the conservativeness of intra-species mi R-92b(bbe-mi R-92a~e).Combining the results of the evolutionary trees of the mi R-92 family of different species,we concluded that mi R-92 b should be a more ancient and conserved gene.2.We analyzed genes potentially targeted by amphioxus miR-92b-5p using GO(gene ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis.It was found that GO terms of these potential target genes enriched in metabolic processes,growth and development and other life activities while KEGG enrichment in the MAPK pathway.It was known that the MAPK pathway could cross talk with the TLR pathway,so it is tempting to speculate that mi R-92 b may be involved in immune regulation.This research provided support for better understanding of the basic information of amphioxus mi R-92 family.3.Using bioinformatics methods(mi Randa and RNA22v2 software)to predict the potential target genes of the mi R-92 family involved in the regulation of TLR signaling pathway,it was found that amphioxus,bbe-miR-92b-5p and bbe-mi R-92d-5p can potentially target the IKK gene in the TLR pathway.4.The dual luciferase reporter assay was used to validate the targeting relationship between the two mi RNAs(bbe-mi R-92b-5p and bbe-mi R-92d-5p)and IKK.The results showed that bbe-mi R-92b-5p could repress luciferase expression via targeting the coding region of IKK gene,while bbe-mi R-92d-5p fails to participate in the regulation of IKK gene.In addition,we further verified the interaction between bbe-mi R-92b-5p and IKK gene by western blot.5.The tissue expression pattern of the bbe-mi R-92b-5p and IKK genes of amphioxus was examined using RT-q PCR,showing that the bbe-mi R-92b-5p and IKK genes are widely expressed the five tissues of gonad,gill,hepatic blind sac,intestine and muscle.Among them,bbe-mi R-92b-5p is highly expressed in gonads and hepatic,but low in muscles;while the IKK gene is low in gonads but high in muscles and gills.Analysis of the expression level of bbe-mi R-92b-5p at different time points(2 h,4 h,8 h,12 h,24 h and 48 h)after LPS stimulation showed that it was significantly up-regulated after stimulation.Following this,we further analyzed the protein level of IKK after LPS stimulation for 12 h,24h and 48 h.The results showed that the expression of IKK protein was down-regulated 12 hours after stimulation,and up-regulated again at 48 hours.6.We also further analyzed the targeting relationship between IKK gene and mi R-92 family across different species,finding that in most species the IKK may be targeted by mi R-92 family.Although there might be false positives,it may to some extent reflect that mi R-92 b may be functionally conservative during evolution.This study lays the foundation for further investigating the function of mi R-92 b in the regulation of immune pathways in amphioxus and other species. |
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