Histone Acetylation Proteins Recruited By Islet-1Promote The Differentiation Of Mesenchymal Stemcells Into Cardiomyocyte-like Cells

Objective To screen and analyze the proteins interact with Islet-1during the differentiation of C3H10T1/2cells transfected withIslet-1lentiviral vector into cardiomyocyte-like cells,and to confirm thepivotal role of Islet-1.Methods1.Transfect of C3H10T1/2cells with Islet-1lentiviral vector andobserve cell morphous in different time.2.Position of Islet-1in cells was detected by immunofluorescechemistry， the protein extraction time was detemined by Western-blot.3.The Islet-1-binding proteins were precipitated by CO-IP.SDS-PAGEseparates and then coomassie brilliant blue staining the proteins.HPLC-CHIP-NanoSpray-ESI-MS/MS screened the proteins.4.Screening proteins at two aspects of histone acetylation and FGFsignal transduction with databases such as ExpAsy、swiss-port.5.Western-blot verified the proteins intacted with Islet-1includingHATs HDACs and FGF Signal transduction proteins.Results1.GFP was detected about in4days after cells been transfected with Islet-1lentiviral vector both in induction group and blank controlgroup.Transfect efficiency of induction group was88.82%and blank groupof90.12%. Cells morphology consistented, showed a fibroblast-like cells,almost norefraction, three-dimensional felt slightly stronger in inductiongroup.while in blank group and C3H10group cells arranged irregular,three-dimensional felt bad;2. The location of Islet-1was in cytoplasm in each group, thefluorescence intensity of induction group was significantly higher than blankgroup and C3H10group(5707.220±174.239.584±6.321,241.377±6.197respectively,P<0.05）. Islet-1in induction group was significant in thethird week (0.782±0.015) compared with the first2week（sP<0.05）, but nodistinction with4weeks（P<0.01）. The expression of Islet-1in inductiongroup was significantly higher than that in blank control group and C3H10group(0.819±0.026,0.127±0.006and0.126±0.001respectively, P<0.05）.3.Western-blot showed that the expression of Islet-1in induction groupwas significantly higher than that in blank control group and C3H10group(P<0.05);we choose7specificity straps to be analyze by MS.4.Bioinformatic analysis showed that parts proteins such as FGF andDNA-binding proteins.5.Verification test results:(1) The synergism of Islet-1was GCN5、P300/CBP、PCAF and HDAC4,HDAC5.(2)FGFSignal transduction-relatedproteins FGF10was alsoin the synergism recruited by Islet-1.Conclusions(1) The synergism of Islet-1and its interacting proteins GCN5、P300/CBP、PCAF and HDAC4、HDAC5played a specific assisting role inthe differentiation of C3H10T1/2cells into cardiomyocyte-like cells;（2）FGF signal transduction-related proteins FGF10and FGF2rb directly binded with Islet-1to regulate C3H10T1/2differentiating intocardiomyocyte-like cells.