Multi-omics Analysis Of Limb Regeneration In Cynops Orientalis

Background:Cynops Orientalis has been established as an ideal animal model for studying limb regeneration.Limb regeneration in salamanders involves many complex biological processes.During different stages of limb regeneration,different genes,signaling pathways,and proteins are activated and synthesized to regenerate lost tissues.Although many studies have been carried out on histology,cytology,biomolecules and signaling pathways of limb regeneration in salamander,the molecular mechanisms of limb regeneration remain largely unclear.Methods:In our study,the regenerating tissue at five different regeneration time points(3 dpa?7 dpa?14 dpa?30 dpa and 42 dpa)post amputation were harvested.RNA-seq?iTRAQ and miRNA-seq technology were applied to study the molecular mechanism of salamander limb regeneration from transcriptomic,Proteomic and miRNA insight.Key transcription factors/proteins/microRNAs that showed differential expressions in the process of limb regeneration were detected,and their potential biological functions in the process of limb regeneration were discussed.The interaction network of differentially expressed miRNAsmRNAs-proteins was constructed by integrated analysis based on transcriptome data and proteomics data.Furthermore,sequence analysis of differentially expressed genes Coro1 a,Capg,Anxa1 and Agr were performed to prepare polyclonal antibodies.The expression and localization of differential genes were examined by using Western blot and immunofluorescence staining.Results:1?Transcriptome analysis of Cynops orientalis regenerating limbA total of 154,790 Unigenes were detected.Compared with the control group,705 differentially expressed genes were screened after redundancy,among which there were up to 539 differentially expressed genes in 7 dpa.These differential genes are involved in 22 biological processes,10 cellular components,and 10 molecular functions,and are assigned to 116 different KEGG pathways.Most of the genes that change significantly during regeneration are closely related to cellular processes,biological regulation,development,and immune system.2.Proteomics analysis of Cynops orientalis regenerating limbWe analyzed the proteomes of limb regeneration on Cynops Orientalis,and 2636 proteins were identified.Of these protein,253 proteins were differentially expressed during limb regeneration.Among these,153 proteins were significantly up-regulated and 87 proteins were significantly down-regulated after limb amputation.The temporal-spatial expressions of 13 proteins showed variable trends.Differential protein GO analysis showed,it was enriched in 23 biological processes,9 cell components and 11 molecular function.Functional analysis indicated that the differentially expressed proteins were associated with metabolism,wound healing,cellular process protein,binding and immune response.3.miRNA analysis of Cynops orientalis regenerating limbA total of 1171 miRNAs,including 534 known and 637 new miRNAs,were identified by using miRNA-seq technology.203 miRNAs were identified to be differentially expressed between the regenerative group and the control group.Together with the proteomic data obtained from our study,integrative analysis of multiple profiling datasets was performed to construct an interaction network of differentially expressed mi RNAs,mRNAs,and proteins.Results of GO and KEGG analyses showed that the differentially expressed miRNAs targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism.The stagespecific regulation of miRNAs on their targets were analyzed by hierarchical clustering analysis and validated by qPCR.The negative regulation of miR-223 and miR-133 a on their targets was tested by performing dual luciferase reporter assay.4?Cloning of key genes during limb regeneration and verification of omics resultsBased on the transcriptome and proteomic data and qPCR verification results,the differential expression genes(Coro1a,Capg,Anxa1 and Agr)were cloned,and interspecies sequence alignment and phylogenetic tree construction were performed.As a result of the expression by western blot,we found the expression levels of the four proteins were significantly up-regulated after amputation.Immunofluorescence results showed that CORO1 A,CAPG and AGR were located in cytoplasm,while ANXA1 were located on cytomembrane,Also,the fluorescence intensity of the proteins were different among five regeneration stages.To sum up,this study initially revealed the regulation mechanism of limb regeneration.Moreover,the integration analysis provides a powerful tool to identify the regulatory miRNAs and their targets during limb regeneration.These results may have strong implications in understanding the complex mechanisms underlying newt limb regeneration.