| Regeneration,the seventh of the]25 most scientifically-challenged scientific issues published by Science,has been the most concerned scientific hotspot.At present,researches on regeneration mainly focuses on the regeneration of animals such as Drosophila,planaria,and pupa and human organs.As the echinoderm,A.japonicus(Apostichopus japonicus)is the group with the highest classification status in invertebrate animals and has superior regenerative ability.It will excrete internal organs in response to the stimulus,and quickly regenerate a complete set of internal organs It is gradually becoming a new model for regeneration research.DNA methylation is one of the earliest found modifications that can cause chromatin structure,DNA conformation and stability,and changes in the way DNA interacts with proteins,thereby controls genes expression,cell reprogramming and stem cell differentiation,and Plays an important regulatory role in organ regeneration.Due to the lack of research methods and basic data,DNA methylation researches lags behind in the regulation of animal specific physiological behaviors in invertebrates,especially in echinoderms.Genome researches that finished by our group has brought a good opportunity for the study about DNA methylation modification role on the regulation of intestinal regeneration of A.japonicus.In summary,studying the role of DNA methylation modification and candidate regeneration genes in intestinal regeneration of sea cucumber not only can provide a reference for the molecular mechanism of intestinal regeneration of A.japonicus,but also provide new ideas for research on human regenerative medicine.At the same time,it will provide theoretical guidance for the research of DNA methylation regenerative drug and regeneration pathways.In this study,WGBS(Whole Genome Bisulfite Sequencing),RNA-Seq,RT-qPCR,Western-blot,and immunohistochemistry were used to study the role of DNA methylation modification and candidate genes in intestinal regeneration of A.japonicus and study expression characteristics and regulatory mechanism of DNA methylase and DNA demethylase during intestinal regeneration of A.japonicus.DNA methylation inhibitors were used to analyze the effect of DNA methylation on intestinal regeneration of A.japonicus.We construct the genome-wide DNA methylation map of intestinal regeneration of sea cucumber,and analyze the DNA methylation regulation characteristics of A.japonicus's intestinal regeneration process.And further we associated DNA methylation with transcriptome analysis results to analysis the control mechanism of A.japonicus intestinal regeneration.We identify the spatio-temporal expression characteristics of key regeneration genes and analyze their regulatory roles.The results of the study are as follows:1.Research on the Regulatory Mechanism of DNA Methylase in Intestinal Regeneration of A.japonicusThe mRNA expression levels of related genes in the DNA methylation regulatory system of A.japonicus were significantly upregulated during the regeneration process,suggesting that DNA methylation modification participates in the intestinal regeneration of A.japonicus.The open reading frame regions of DNA methylation key regulatory enzymes Dnmt3b,TDG and Tet2 were obtained by cloning.After the constructed three key regulatory enzymes overexpression vectors and interfering fragments in vitro were transfected,the expression of proliferation related genes were upregulated.2.DNA methylation can affect Intestinal Regeneration of A.japonicusThe primary culture system of intestinal cells of A.japonicus was successfully constructed.High-level inhibitors inhibited cell survival.Inhibition of DNA methylation inhibited the proliferation of primary cells of A.japonicus and intestinal regeneration in vitro and in vivo,indicating that the key role of DNA methylation modification in intestinal regeneration of A.japonicus.In the process of DNA methylation inhibition,most mRNAs of proliferation-related genes were up-regulated.3.Construction of DNA methylation map in intestine of A.japonicusDuring the regeneration process,the differential DNA methylation-related genes are mainly CG methylation types,and are mainly distributed in the middle and the latter half of exon region of the coding region of the gene.KEGG analysis showed that the differential DNA methylation genes were significantly enriched in neural,immunological,metabolic pathways,and ubiquitylation and phosphorylation modifications.At the same time,DNA methylation of RIPK1,FGFR3,KRAB1,Notch1,and ROB04 genes during intestinal regeneration was verified..DNA methylation level of RIPK1 and KRAB1 were lower,and DNA methylation level of FGFR3 were Higher at 2 h in intestinal regeneration;DNA methylation level of Notch1 and ROB04 were also higher at 3 d.4.Construction of transcription expression profile at early stage of intestinal regeneration of A.japonicusThe transcriptional expression profile of the primary stage of A.japonicus regeneration was constructed.The results showed that the significantly up-regulated genes in the primary regeneration stage were mainly enriched in the immune and neural related pathways,and were consistent with the DNA methylation genes clustering results.The results of DNA methylation and transcriptome combined analysis showed that the expression pattern of DNA methylation regulation in different genes was different.Most methylated genes inhibited genes transcription and DNA demethylation genes promoted genes transcription and expression.Most related genes to phosphorylati were DNA methylated in the gene's body regions at 2 h and their expressions were down-regulated.Most of the phosphorylation-related genes had DNA methylation in the promoter region and their expressions were up-regulated at 3 d.Expression patterns of other classified genes were differences in different genes.5.Regulatory Mechanism of Regeneration Candidate Genes in Intestinal Regeneration of A.japonicus(1)The open reading frame sequences of KRAB1,FGFR3,ROBO4,and RIPK1 genes that regulated by DNA methylation were detected,and they were up-regulated expression during the intestinal regeneration.After transfecting constructed KRAB1,FGFR3,ROBO4 and RIPK1 overexpression vectors in vitro,we found KRAB1 inhibited cell proliferation,and FGFR3,ROBO4 and RIPK1 promoted cell proliferation and related genes expression.(2)We obtained the full-length sequence of WntA from A.japonicus and analyzed its expression characteristics.The results showed that WntA expression gradually up-regulated during regeneration,WntA protein localized in the epidermal cells,intestinal muscle and submucosa,and expression patterns are similar with cell proliferation localization.(3)Catenin? and DVL3 in the canonical Wnt signaling pathway,and PKC in the non-canonical Wnt/Ca+ pathway and other Wnt signaling pathway-related genes expression were significantly up-regulated on the 21st day of intestinal regeneration. |
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