Effect Of CSRP2BP On Differentiation Of Human Embryonic Stem Cells Into Trophoblast Cells And Apoptosis

Objective:To study the effect of histone acetyltransferase CSRP2BP on the differentiation of human embryonic stem cells into trophoblast cells,and to find out the correlation between CSRP2BP and pregnancy-related diseases caused by trophoblast cells.At the same time,combined with the results of microarray,the mechanism of CSRP2BP on the apoptosis of H1 cells was explored.Methods:CSRP2BP silenced in the H1 cell line was constructed by lentiviral transfection,and this cell line was induced to the trophoblast cells to observe the changes of cell morphology.The immunofluorescence,the HCG content of the cell supernatant and the qPCR method were used to compare the change of silent group and silencing control group.Analysis of silencing CSRP2BP specifically affects which type of trophoblasts H1 cells differentiate into.The gene expression microarray technique was used to analyze the changes of gene expression profile of H1 cells after silencing CSRP2BP,and to screen out the endogenous CSRP2BP and apoptosis-related genes and their possible target genes.Flow cytometry was used to verify the apoptosis of H1 cells silencing CSRP2BP.Results:Two H1 cell lines which silencing CSRP2BP were successfully constructed,and the morphological changes of cytotrophoblastic cells were observed on the 3rd day after induction.The trophoblast-like cell morphology was more obvious with the induction time,and the silencing control group showed obvious nourishment on the 6th day.In the layered cells,the silent group showed significant trophoblast-like cells on day 9.The HCG value of the H1shbpCON supernatant of the silencing control group was 1320.71 mIU/ml,and the HCG value of the supernatant of the experimental group H1shbp1#2 was 12.62 mIU/ml;The HCG value of the supernatant of the experimental group H1shbp3#8 was 14.24 mIU/ml;the expression was significantly different(P<0.001);the marker antibody CK7 of trophoblast cells was detected by immunofluorescence method,and the positive cells rate of CK7 was counted.The silencing control group was 25.2%in H1shbpCON;11.5%in the experimental group H1shbp1#2;7.7%in the experimental group H1shbp3#8,p<0.05,the results were statistically significant.qPCR results showed two groups of trophoblast-related markers(PGF,synthin,HCG-A,HCG-B,H1A-G and GATA3)expression were all increased,but the increase of the silencing control group was higher than that of the silenting CSRP2BP group.The increase rate of the markers,HCG-A and HCG-B,of the somatic trophoblast cells is greater than that of the trophoblastic cell marker H1A-G.The results of the microarray showed that among the genes related to apoptosis,compared with the silenting control group,CDK1,PITX2,and PCSK9 were higHly expressed in the silencing CSRP2BP group,while FAS,FGF,FOS,GADD45,DDIT3,and VEGFA were lowly expressed.The results of flow cytometry showed that the apoptosis rate(early apoptosis and late apoptosis)was increased in the silent group compared with the silenting control group(P<0.05).Conclusions:Endogenous silencing of CSRP2BP inhibits the differentiation of H1 cells into trophoblast cells,which affects the differentiation of H1 cells into syncytiotrophoblasts more than the differentiation of extravillous trophoblasts.Silencing CSRP2BP promotes apoptosis of H1 cells,which may be related to the promotion of apoptosis-related genes CDK1,PITX2,PCSK9,FAS,FGF,FOS,GADD45,DDIT3,and VEGFA.