The Role And Regulatory Mechanism Of MEGF6,KLF13 And BTF3L4 In Intestine Regeneration Of Sea Cucumber Apostichopus Japonicus

Sea cucumber,Apostichopus japonicus is a kind of echinoderm with economic value in the Yellow Sea and Bohai Sea.When it is affected by harmful stimulation,attack and adverse natural environment,it will appear a special defense phenomenon-"spit out the viscera",that is,most of the digestive tract and its connected tissues and organs(respiratory tree,gonad,etc.)will be discharged from the body.When the environment is suitable,a complete set of viscera will be regenerated.However,if the environmental conditions continue to be adverse,or the young spits,it often causes a lot of deaths,which will cause economic losses in aquaculture.The special characteristics of sea cucumbers have also attracted the attention of the field of regenerative medicine.There have been some reports about the molecular regulation mechanism of regeneration in sea cucumber,but there are no reports about the regulatory mechanism of MEGF6,KLF13 and BTF3L4 in the regeneration process of sea cucumber.MEGF6 is a multiple epidermal growth factor,which has similar structural and functional characteristics with EGF,and generally has more than 30 EGF-like domains.MEGF6 plays a vital role in promoting the development and maintaining the stability of the internal environment of the organism,mainly by regulating the growth and migration of biological cells.KLF13 is a basic transcription factor,widely distributed in biological cells.Its C-terminal is a DNA binding domain,which contains three conserved Cys2 / His2(C2H2)zinc finger structures.KLF13 combines the DNA sequences in the promoter and enhancer regions of target genes through the zinc finger structures,which contain the rich CACCC or GC boxes,so as to activate transcription expression and play the role of regulating cell proliferation,cell differentiation,cell cycle process and other functions.BTF3 is a basic transcription factor with a typical NAC domain.BTF3 is widely found in many animals,plants and microbes,and has a dual identity: ? Belonging to the basic transcription factor.It combines with RNA polymerase II,and involves in the control of gene transcription.? Belonging to the NAC family.It prevents misfolding by binding with proteins,and regulates protein synthesis and degradation.In this paper,sea cucumbers were used as the experimental materials to construct the transcriptome of intestine regeneration for 5 days and 10 days by using high-throughput sequencing technology(bgiseq-500 platform).The full-length of Aj-megf6,klf13 and btf3l4 genes were cloned by RACE technology.The subcellular localization of Aj-MEGF6,KLF13 and BTF3L4 were determined by IF technology.The m RNA expression of Aj-megf6,klf13,btf3l4,egfr and wnt6 genes were analyzed by q PCR technology.The expression patterns of Aj-MEGF6,KLF13,BTF3L4 and the related proteins of signaling pathways(EGFR,SNAI2,CDH1,CREB1,CDK2,Cyclin D1,WNT6,BMI1)in the regeneration process of intestine were analyzed by WB technology,and the molecular mechanism was studied.The research of Aj-MEGF6,KLF13 and BTF3L4 in the regeneration process is of great theoretical significance for the exploration of the regeneration mechanism of sea cucumber,and has potential application value for preventing or avoiding the death or reduction of production caused by spitting in the process of seed cultivation and cultivation of sea cucumber.The experimental results of this study were as follows:1.The results showed that there were 43811752 clean reads,42987312 clean reads and43507122 clean reads in the control group,5 days regeneration group and 10 days regeneration group,respectively,with an average mapping rate of 44.35% in the genome and 28.36% in the gene set.The 27766 genes were detected.Compared with the control group,5255 genes were upregulated and 3849 genes were downregulated in 5 days regeneration group;4179 genes were upregulated and 4780 genes were downregulated in 10 days regeneration group.Megf6,klf13 and btf3l4 were upregulated significantly in both 5days and 10 days regeneration groups.2.The full length c DNA of Aj-megf6 was 5665 bp,which encoded 1604 amino acids and37 EGF like domains.It was predicted that the molecular weight of Aj-MEGF6 protein was172.5 k Da,being a secretory protein,which had a signal peptide.It was a hydrophilic and non-transmembrane protein,containing 53 phosphorylation sites.The result of localization prediction indicated that 52.2% of Aj-MEGF6 was located in the nucleus,17.4% in the mitochondria,8.7% in the cell membrane,8.7% in the cytoplasm,4.3% in the vesicles of secretory system,4.3% in the golgi body and 4.3% outside the cell.According to the sequence alignment of MEGF6,it indicated that the sequence similarity between Aj-MEGF6 and that of 18 other species was 38.59 ? 49.07%,with maximal similarity between AjMEGF6 and that of sea urchin Strongylocentrotus purpuratus.The result of NJ phylogenetic tree showed that the three species of echinoderm(A.japonicus,S.purpuratus and Acanthaster planci)were clustered into a cluster,which was in line with the classification and evolutionary status.The full length c DNA of Aj-klf13 was 2496 bp,which encoded 284 amino acids and three C2H2 domains.It was predicted that the molecular weight of Aj-KLF13 protein was 32.5k Da.Aj-KLF13 was not a secretory protein,which had no signal peptide.It was a hydrophilic and non-transmembrane protein,containing 26 phosphorylation sites.The result of localization prediction indicated that 95.7% of Aj-KLF13 was located in the nucleus,4.3% in the vacuolar.According to the sequence alignment of KLF13,it indicated that the sequence similarity between Aj-KLF13 and that of other 19 species was 35.64 ? 49.83%,with maximal similarity between Aj-KLF13 and that of sea urchin S.purpuratus.The result of NJ phylogenetic tree showed that the three species of echinoderm(A.japonicus,S.purpuratus and A.planci)were clustered into a cluster.The full length c DNA of Aj-btf3l4 was 1099 bp,which encoded 161 amino acids and a NAC domain.It was predicted that the molecular weight of Aj-BTF3L4 protein was 17.9k Da.Aj-BTF3L4 was not a secretory protein,which had no signal peptide.It was a hydrophilic and non-transmembrane protein,containing 5 phosphorylation sites.The result of localization prediction indicated that 56.5% of Aj-BTF3L4 was located in the nucleus,26.1% in the mitochondria,13.0% in the cytoplasm,4.3% in the vesicles of secretory system.According to the sequence alignment of BTF3L4,it indicated that the sequence similarity between Aj-BTF3L4 and that of other 14 species was 54.55 ? 58.43%,with maximal similarity between Aj-BTF3L4 and that of sea urchin S.purpuratus.The result of NJ phylogenetic tree showed that sea cucumber A.japonicus and sea urchin S.purpuratus were clustered into a cluster.3.The expression patterns of Aj-megf6,klf13,btf3l4,egfr and wnt6 m RNA levels in the process of intestine regeneration showed a similar trend,that was,they first increased and then decreased.The peak values were reached at 6 ? 12 days(126.47,53.36,4.19,5.32 and8.32 times of the control group),and the expression decreased to the level of the control group at 15 ? 21 days.4.According to the localization results of IF,Aj-MEGF6 was mainly distributed in cytoplasm and extracellular region,which was different from that of bioinformatics prediction,being mainly located in nucleus.Aj-KLF13 and Aj-BTF3L4 were mainly distributed in nucleus,which was consistent with that of bioinformatics prediction.5.The expression patterns of Aj-MEGF6,KLF13,BTF3L4,EGFR and WNT6 proteins were similar to those of m RNA levels,and they first increased and then decreased.The peak values were reached at 6 ? 12 days(9.32,1.64,1.26,3.89 and 3.59 times of the level of the control group),and the expression decreased to the level of the control group at 15?21 days.The expression patterns of SNAI2,CREB1,CDK2,Cyclin D1 and BMI1 in the process of intestine regeneration were basically the same as that of Aj-MEGF6,KLF13 and BTF3L4 proteins,reaching the peak value at 6?12 days,then decreasing gradually,respectively.However,the change trend of CDH1 was the opposite,that was,it first decreased and then increased,reaching the minimum at 6 days.6.MEGF6 was one of the main components of ECM,which was responsible for maintaining the integrity of the organization.In the process of intestine regeneration,AjMEGF6 and EGFR were highly expressed,which could promote the combination,and Snai2 was upregulated by TGF ? / Smad signaling pathway.Snai2 could promote Cyclin D1 expression,and wnt6 overexpression could also cause Cyclin D1 upregulated expression with the participation of kinase,which contributed to cell proliferation.KLF13 was an effective activator of Cyclin D1 promoter,and the activation process could be enhanced by GATA4 to promote Cyclin D1 transcription expression.At the same time,CREB could combine with CRE sequences in Cyclin D1,and promoted gene transcription and upregulated expression of Cyclin D1.CDKs could combine with Cyclin to formheterodimer,which could catalyze the phosphorylation of different substrates through CDK activity,thus they could promote cell cycle.At the same time,SNAI family was also one of the inhibitors of CDH1.When the expression of SNAI2 was high and CDH1 was inhibited,EMT would be induced to promote the proliferation and migration of cells.As the upstream gene of BMI1,BTF3 could inhibit the ubiquitination and degradation of BMI1.As a key regulatory factor,which could control the pluripotency and early development of embryonic stem cells,BMI1 could mediate Wnt signaling pathway,and also promote EMT,controlling cell self-renewal.The mesenchymal cells had the potential of self-renewal and multidirectional differentiation,and were the basis of normal development and wound healing.The mesenchymal cells in mesentery thickening provided a cell source for the intestinal cavity regeneration of sea cucumber,and the cell division and proliferation promoted the regeneration and reconstruction of intestinal tissue in sea cucumber.