Effects And Mechanisms Of SUMOylation Of Foxm1 On Pluripotent Maintenance Related Genes, Pathways And Cell Proliferation In Stem Cell Liked Cell

Fork-head box transcription factor M1(Foxm1)is an indispensable factor that regulates the DNA damage response,the G2/M phase transition and mitosis.Additionally,it plays an essential role in regulating embryonic development and pluripotent maintenance.Small ubiquitin-like modifier(SUMO)is widely found in eukaryotic cells.As a kind of post-translational modification,SUMOylation alters stability,subcellular localization and interaction with other proteins of substrates.The studies show that in several kinds of carcinoma cells,SUMO1 can be able to modify and change biological function of Foxm1.In order to illustrate the effects and mechanism of SUMOylated Foxm1 in stem cell,F9 teratoma cell line is introduced as model.Meanwhile,experiments such as overlap extension PCR,Western blot,immunoflourescence,real time-qPCR and double luciferase report system are used to reveal the effects of Foxm1 SUMOylation in F9.The results of this research are as follows:1.In this research,an in-silico prediction algorithm is used to predict the SUMOylation motifs in Foxm1.There are seven motifs are recognized by such algorithm.Abstracting total RNA from F9 cell and copy the CDS sequence of Foxm1 from it.Then,inserting this sequence into pCMV-Myc to generate a eukaryotic expression vector pCMV-Myc-WT-Foxm1.Based on that,mutating seven motifs successively to generate a mutant that cannot be SUMOylated(Foxm1-7KR).After that,we copy the CDS sequences of SUMO1 and Ubc9 and insert them into p CMV-HA to generate pCMV-HA-SUMO1 and pCMV-HA-Ubc9.Finally,the results of Western blot show that each of seven motifs in Foxm1 can be modified by SUMO1 respectively.When all motifs are mutated,Foxm1 cannot be SUMOylated.2.We copy and insert the promoter region of Oct4 into pGL4.0 to generate pGL4.0-Oct4 p.After co-transfecting Foxm1,SV40 and pGL4.0-Oct4 p with or without SUMO1 and Ubc9 to F9,we exert double luciferase report analysis.It shows that SUMOylation inhibits the activation effect of Foxm1 on Oct4.Besides,RT-qPCR reveals that SUMOylation of Foxm1 also inhibits trranscription of Oct4,Sox2 and Nanog.To find out the mechanism of these phenomenon,we add 10?g/ml cycloheximide into culture medium to inhibit protein synthesis in the cells.The results of western blot show that SUMOylated Foxm1 are less stable.Furthermore,we exert immunoflourscence to demonstrate that unSUMOylated Foxm1 are mainly accumulated in the nuclear of cells while SUMOylated Foxm1 are distributed in both nuclear and cytoplasm.3.Copying and exerting the sequences of WT-Foxm1 and Foxm1-7KR in lentivirus vector pCDH-MCS-T2A-Puro-MSCV to generate two kinds of plasmid.By using these plasmids to screening the F9 cell lines that highly express WT-Foxm1 or Foxm1-7KR.Then CCK8 and cell cycle analysis are introduced to reveal the influence of SUMOylation of Foxm1 on cell proliferation and cell cycle.It shows the cell line that highly express WT-Foxm1 proliferates slower than the cell line highly express Foxm1-7KR.And SUMOylation of Foxm1 causes S phase arrest.4.We purchased the double luciferase report vector which contain the promotor sequences of ?-catenin: Super TOPFlash.Co-transfecting Foxm1,SV40,Super TOPFlash with or without SUMO1 and Ubc9 to F9 to illustrate the influence of SUMOylated Foxm1 on Wnt/?-catenin pathway--an essential pathway to maintain the pluripotency of stem cells.Double luciferase report analysis demonstrates that SUMOylation inhibits the activation effect of Foxm1 on ?-catenin.Also,the outcomes of immunoflourscence and Western blot conformably show that SUMOylation of Foxm1 prohibits the nuclear entrance of ?-catenin.Finally,we use Co-immunopricipitation to prove that one of mechanisms of such phenomenon is SUMOylation weaken the interaction between Foxm1 and ?-catenin.