The Regulation Of Nspc1 On The Differentiation Of P19 Cells Into Cardiomyocytes

Objective: Polycomb group protein(PcG) is a kind of highly conserved and widely existed protein, which plays an important role in the development and maintenance of embryonic stem cells. Previous study in P19 cells suggests that Nspc1 plays a positive role in the regulation of Oct4、Sox2 and Nanog maintenance genes. Treated with dimethylsulfoxide(DMSO), P19 cells can be induced to cardiomyocytes, and Nspc1 decreased firstly and then increased, Ch IP with DNA Micorarray indicated that Nspc1 has binding sites beside promoters of myocardial differentiation mark gene Nkx2.5, Mef2 c, Hand2 etc. Therefore, the study on the role of Nspc1 in the differentiation of stem cells into cardiomyocytes is helpful to reveal the molecular mechanism of the regulation of cardiac differentiation.Methods: 1、The relationship between Nspc1 and tissues、cell lines.â‘ RT-PCR and Western blot are used to analyze the expression of Nspc1 in different tissues and cell lines.â‘¡With P19 cells, for example, change the expression of Nspc1 by transfection, use immunofluorescence and flow cytometry to identify Nspc1 protein subcellular localization and its effect on cell cycle.2、Analyze the role of Nspc1 in regulation of target genes in DMSO differentiation cell model of P19 cellsâ‘ Detect key factors in myocardial differentiation model by RT-PCR / Real-time PCR and Western blot.Analyz the changes of Nspc1 and cardiac marker gene RNA and protein expression level.â‘¡Analyze the effects on myocardial differentiation model about main control genes and myocardial marker genes after change Nspc1 gene level: in DMSO induced P19 cells,we transfected with plasmid or siRNA at different time points(0h, 6h, 12 h, 24 h, 48 h, 72h) in order to overexpress and knock down Nspc1,then monitor the cardiac differentiation mark genes expression(Nkx2.5 and Gata4/6 and Mef2c) and speculated the downstream of Nspc1 target genes.3、The regulation mechanism of target genes in the differentiation model of P19 by Nspc1â‘ Predict the sequence Nspc1 combined with and Ch IP assay analysis: we used bioinformatic software to analyze the promoter structure of the target genes, with undifferentiated P19 cells, anti-Nspc1 antibody and anti-H2 A ubiquitination antibody were used,too. ChIP with PCR was used to explore objective protein binding sequence so as to abtain target gene promoter information and epigenetic modification state analysis.â‘¡Target gene promoter report gene vector construction and analysis of transcriptional activity :we used molecular cloning methods from the mouse genome to clone target gene promoter sequences, and cloned into the pGL3 basic luciferase report gene vector. Over-expressing the Nspc1 gene in P19 cells, we uesd mammalian cell Dual-Luciferase®Reporter Assay System to detect the differences in the transcriptional activity of Nspc1 during the process in regulating target gene, mutations start activity and confirmed the main effect of Nspc1 in target genes and the precise regulatory sites.Results:1、Nspc1 is highly expressed both in brain and myocardial tissue of adult rats. In different cell lines, the expression of Nspc1 in P19 cells was the highest(P<0.05). In addition, Nspc1 was mainly expressed in the cell nuclei by immunofluorescence technique. The results indicated that the S phase increased and G2 phase decreased(P<0.01).2、In the course of P19 cell differentiation, the marker gene increased gradually with time. The stem cells pluripotency factors were all decreased, Nspc1 decreased first and then increased. The difference was statistically significant.After transfection, P19 cells were cultured 0-72 h, in this process,pluripotency factors、myocardial marker genes and Nspc1 expression changes were analyzed. The results showed that with the increase of Nspc1, Mef2 c and Gata4 inhibition is more significant. The difference was statistically significant.3、With ChIP and PCR methods,we found that in normal P19 cells, Mef2 c promoter region-601bp~-180 bp,the ubiquitination levels of histone H2 A compared with the control group were much higher. When Nspc1 mAb bound increased, the corresponding ubiquitination level of histone H2 A was improved,but Gata4 promoter in the promoter region of histone H2 A ubiquitination level was lower than the control group. From spatial view(H9c2 cells): the u H2 A level in the CHIP5 region(-601/-180) was higher, and also with the presence of Nspc1 binding. The difference was statistically significant.4、Using molecular cloning and Dual-Luciferase®Reporter Assay, the results showed that the luciferase activity of recombinant vector in P19 cells was significantly enhanced. At the same time, Mef2c-0.8kb had higher transcriptional activity(P<0.001). In addition, the luciferase activity of the three luciferase report gene plasmids increased with the increase of Nspc1 gene expression. The difference was statistically significant.Conclusion: Oue work reveals a new target gene transcriptional regulation pattern of PcG family protein Nspc1, which provides clues for us to further study the regulation mechanism in the process of differentiation of cardiac muscle cells from a derivative of P19 embyonal carcinoma cells.