| 其他摘要 | Regeneration, the seventh of the 125 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. japonicus
The mRNAexpression 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. DNAmethylation can affect Intestinal Regeneration of A. japonicus
The 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 DNAmethylation map in intestine of A. japonicus
During 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 ROBO4 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 ROBO4 were also higher at 3 d.
4. Construction of transcription expression profile at early stage of
intestinal regeneration of A. japonicus
The 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) Cateninβ 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.
Key words: Apostichopus japonicus, Regeneration, WGBS, DNA methylation,
WntA |
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