Molecular Mechanism Of SP600125-induced Cell Polyploidization In Fish

Previous studies have demonstrated that SP600125 could induce polyploidy and a stable autotetraploid cell line from diploid crucial carp caudal fin cells has been obtained.In order to explore the molecular mechanism of SP600125 inducing cell polyploidization in fish.This study makes use of the technology of transcriptome sequencing to analyze and compare the differential gene expression profiles between SP600125-treated and control cells,while using GO and KEGG pathway enrichment analysis to comprehensively explore the signal regulatory networks and key functional genes involved in SP600125-induced cell polyploidization and cell homeostasis maintenance.It aims to reveal the mechanism of SP600125 inducing polyploidy and the mechanism for maintaining stability or reconstructing cellular homeostasis as well as providing theoretical basis and technical reference for the application of SP600125 in fish developmental biology research and polyploidy cultivation.The specific results are as follows:1.Analyzing transcription profile level of SP600125 treated cells by high throughput sequencingThrough transcription sequencing and comparative analysis of SP600125 treated diploid crucial carp caudal fin cells and control in vitro were carried out by high-throughput sequencing technology.We obtained 96,997 unigenes and performed GO function annotations and enrichment analysis of KEGG pathway.Compared with the control cells,a total of 4516 differential expression genes in among2670 genes were up-regulated and 1846 genes were down-regulated of cells treated by SP600125.And the gene functional clustering analysis of KEGG the differential expression genes were mainly related to cell metabolism,stress response,development process and cell cycle regulation.The results showed that SP600125 could change the expression levels of a number of functional genes related to cell proliferation regulation,especially those related to cell cycle checkpoint.All of the above demonstrated that SP600125’s multiple regulation of cell cycle checkpoint is closely related to the reglulation of fish cell polyploidization induction.2.Regulatory effect of p53 signal pathway on cell polyloidization induced by SP600125Based on the results of transcriptomics analysis,we further identified the role of p53 signaling pathway closely related to cell cycle checkpoint in SP600125 induced cell polyloidization.According to the established SP600125 interval cycle induction polyloidization strategy,the effects of SP600125 on the mRNA and protein levels of related genes in the p53 signaling pathway were detected by qRT-PCR and Western blot technologies.The results showed that SP600125 could induce a significant up-regulation of mRNA expression levels of functional genes related to cell cycle regulation,such as p53,Mdm2,p21 and Gadd45ɑ.qRT-PCR results is consistent with the results of transcriptome analysis.Western blot results also showed that the protein expressions of p53,MDM2 and p21 were significantly up-regulated after 48h of SP600125treatment.In order to further verify the role of p53 pathway in inducing polyploid cells by SP600125,cells were incubated with p53 inhibitor PFT-ɑand the results showed that PFT-ɑtreatment could reduce the cell cycle arrested G₂/M phase to a certain extent and the production of polyploid cells under SP600125 induction conditions.The above results show that p53 pathway is involved in the production of polyploidy induced by SP600125 via the regulation of cell cycle arrested G₂/M phase.3.Effect of spindle assembly checkpoint（SAC）regulated genes on cell polyloidization induced by SP600125Cytological observations showed that SP600125 treatment caused the cells to be arrested mitotic phase.Transcriptome analysis and qRT-PCR results showed that SP600125 resulted in downregulation of cell CyclinBl/3,Cdc2,Securin and spindle assembly checkpoint（SAC）related genes Bub1,Mad2 and Cdc20 mRNA levels responsible for kinetochore adhesion and chromosome segregation monitoring.Western blot results further confirmed that SP600125 induction decreased BUB1,MAD2 and CDC20 protein expression levels in cells.Adenovirus was used to successfully construct the overexpression recombinant vector（pAd-mCMV-MAD2-3-Flag-GFP-pA）of SAC core protein MAD2.Combined with qRT-PCR,Western blot and flow cytometry analysis,the effect of overexpression of MAD2 on SP600125induced cell polyloidization were further identified.The results confirmed that overexpression of MAD2 could significantly reduce polyploid cell generation caused by SP600125 interval cycling treatment.The above results showed that SP600125 induced the decrease of activity of CyclinB1-Cdc2 complex and spindle assembly checkpoint（SAC）,which led to mitotic slippage and endomitosis induce cell polyloidization.4.Cells homeostasis regulation participated by acute immune which under the stress of SP600125Based on transcriptome sequencing analysis,diploid crucian carp caudal fin cells treated with SP600125 cause significant changes in the expression level of a large number of immune inflammation-related genes,which are involved in innate defense,inflammatory pathways and cell adhesionmolecule-relatedpathways,includingproinflammatory cytokines（such as IL-1β,TNF-ɑand IL-6R）,transcription factor signal transduction molecules（such as Stat-1/3 and IκB）,cell adhesion molecules（such as Vcam-1）and integrin’s（such asɑ2β1andɑMβ2）etc.At the same time,SP600125 treatment resulted in significant changes not only in morphology and numbers of mitochondria but also in mRNA levels of Glut1 and major glycolytic enzyme genes（such as Hk1,Pfbfk,Pgam,Eno and Ldh）,and mRNA levels of mitochondrial electron transport chain genes（such as NADH dehydrogenase Nd1,Nd2 and Nd5complex I subunit gene,Adh,Ndufs8,cytochrome C related genes Ndufa4 and Uqcr and Sdhd）were also significantly reduced.Relevant research results reveal that cells participate in the maintenance of cell homeostasis through stress immune response and reduction of oxidative phosphorylation level under SP600125 stress.Conclusions:（1）Transcriptomic analysis shows that the multiple regulation of SP600125 on cell cycle checkpoints is closely related to the induction of fish cell polyploidization.（2）SP600125 induces G₂/M phase arrest of p53 signal pathway and leads to miotic slippage and endomitosis caused by signal attenuation at spindle assembly checkpoint（SAC）,thus realizing fish cell polyploidization.（3）Under the stress of SP600125,cells participate in the maintenance of cell homeostasis through stress immune response and reduction of oxidative phosphorylation levels.The research results laid a theoretical foundation for the application of SP600125 in fish developmental biology research and production practice.