PHA-mediated MicroRNA Expression Profile Of Lampetra Japonica And Prediction And Verification Of Key Signal Molecules Targeted MiRNA

Lamprey is the oldest representative of jawless vertebrates and the best animal model for studying the origin and evolution of vertebrates.In recent years,more and more researches on the composition of the lamprey's immune system and its regulatory mechanism.Micro RNAs(miRNAs)have been shown to regulate the post-transcriptional expression of metabolism-related genes in a variety of organisms,but the current researches on miRNAs are mostly focused on higher vertebrates,while the research on miRNAs in lampreys is very few.This study took the Lethenteron japonicum as the research object and used high-throughput sequencing technology to perform small RNA sequencing on the supranural myeloid bodies of the lamprey stimulated by phytohemagglutinin(PHA).Lamprey species-specific miRNA precursors were identified by microarray chip analysis.The target miRNA of the key signal molecules in the TCR signaling pathway of higher vertebrates were predicted by using bioinformatics methods in lampreys.The possible miRNA-m RNA targeting regulation mechanism in regulating the immune response of lampreys under the stimulation of PHA provides a basis for subsequent studies on the role of lamprey miRNA.The main results of this study are as follows:1.Using high-throughput sequencing technology,the small RNA sequencing data of lamprey supranural myeloid bodies after PHA stimulation was obtained.Through the establishment of a small RNA database,quality control of the sequencing data,comparison with reference sequences,and analysis and processing of miRNA classification and annotation,a total of 5 974 734 small RNA reads(Read)were finally obtained.According to statistics,a small number of miRNA families exhibit extremely high abundance expression in lamprey supranural myeloid bodies after PHA stimulation.2.For small RNA fragments not annotated in miRBase,the prediction model independently constructed by BGI was used to predict species-specific miRNAs and their precursors through comparing with the sea lamprey database.Totally 56 mature lamprey species-specific miRNA sequences and their precursors were predicted.3.Through customized gene chip microarray analysis,it was discovered and verified that35 species-specific miRNA mature sequences were expressed in lamprey supranural myeloid bodies after PHA stimulation,indicating that these 35 species-specific miRNAs are new members of some miRNA families unique to lampreys.4.The miRDB website was used to predict the miRNAs of humans,mice and chickens that possiblely target at those molecules in lamprey homologus with the key signal molecules of TCR and BCR signaling pathways.Using the program Clustal X,the above prediction results were compared with the entire sequenced lamprey miRNA database,and the lamprey miRNAs homologous to those predicted miRNAs of humans,mice and chickens were found.The RNA22.v2 software was used to verify the binding sites of the 3'UTR region of the homologous signal molecule c DNA and the predicted lamprey target miRNAs.Finally,we predicted that Lja-miR-182-5p may target Lja-Lyn molecules,and Lja-miR-301a-3p may target Lja-Vav2 molecules.5.The expression of those predicted miRNAs and their targets in lamprey supranural myeloid bodies after PHA stimulation were detected by real-time fluorescent quantitative PCR.The results show that the expression level of Lja-miR-182-5p is down-regulated significantly relative to control group(P<0.01),and the expression of Lja-Lyn was significantly up-regulated(P<0.05),which was statistically significant.The expression of Lja-Vav2 was up-regulated,and the expression of Lja-miR-301a-3p was extremely down-regulated,which was statistically significant significance(P<0.01).In summary,in lamprey supranural myeloid bodies,the expression levels of Lja-miR-182-5p,Lja-Lyn,Lja-miR-301a-3p and Vav2 are negatively correlated after PHA stimulation,respectively.We speculate that Lja-miR-182-5p and Lja-Lyn,Lja-miR-301a-3p and Lja-Vav2 may have a targeted regulation relationship.The targeting mechanism between them also requires subsequent in vitro dual luciferase reporter gene analysis and in vivo knockdown and overexpression experiments to verify.