Abstract:

Objective To investigate the effect of paired box transcription factor 3 （PAX3） gene silencing on the differentiation of the P19 cells into the cardiomyocyte-like cells，and to clarify its possible mechanism. Methods Dimethylene maple （DMSO） was used to induce the differentiation of the P19 cells into cardiomyocyte-like cells， and the morphology of the cells during the induction process was observed. The cells were collected on the 0th day （0 d group） and 10th day （10 d group） after induction and differentiation.The P19 cells were infected with sh-PAX3 Lentivirus.The cells were divided into control group，sh-NC group（negative control group）and sh-PAX3 （PAX3 interference）.Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to verify the positive expression of cTnI in P19 cells silenced with PAX3 gene. The P19 cells infected with lentivirus were treated with Notch signal agonist Jagged1 and were induced and differentiated with DMSO for 10 d. The cells were divided into DMSO group， DMSO+sh-NC group， DMSO+sh-PAX3 group， DMSO+Jagged1 group and DMSO+sh-PAX3+Jagged1 group. RT-qPCR method was used to detect the expression levels of GATA binding protein 4 （GATA4）， atrial natriuretic peptide （ANP）， cardiac troponin （cTn）T，PAX3，Notch1， Notch intracellular domain （NICD）， and Hes family bHLH transcription factor 1 （Hes1） mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of PAX3，Notch1， NICD， and Hes1 proteins in the cells in various groups； immunofluorescence assay was used to detect the positive expression of cTnI in the cells the differentiation rates of the cardiomyocyte-like cells in various groups. Results On the 10th day， after induction and differentiation，the cluster of cardiomyocyte-like cells with spontaneous rhythmic contraction could be observed.The RT-qPCR and Western blotting results showed that after lentivirus infection， compared with control group and sh-NC group， the expression levels of PAX3 mRNA and protein in the P19 cells in sh-PAX3 group were decreased （P<0.05）， indicating that the P19 cells silenced with PAX3 gene were successfully constructed. The RT-qPCR results showed that compared with 0 d group， the expression levels of GATA4， ANP， and cTnT mRNA in the cells in 10 d group were increased （P<0.05）， while the expression levels of PAX3，Notch1， NICD， and Hes1 mRNA were increased （P<0.05）； compared with DMSO group and DMSO+sh-NC group， the expression levels of GATA4， ANP， and cTnT mRNA in the cells in DMSO+sh-PAX3 group were decreased （P<0.05）， while the expression levels of GATA4， ANP， and cTnT mRNA in the cells in DMSO+Jagged1 group were increased （P<0.05）； compared with DMSO+sh-PAX3 group， the expression levels of GATA4， ANP， and cTnT mRNA in the cells in DMSO+sh-PAX3+Jagged1 group were increased （P<0.05）. The Western blotting results showed that compared with 0 d group， the expression levels of PAX3， Notch1， NICD， and Hes1 proteins in the cells in 10 d group were increased （P<0.05）； compared with DMSO group and DMSO+sh-NC group， the expression levels of Notch1， NICD， and Hes1 proteins in the cells in DMSO+sh-PAX3 group were decreased （P<0.05），while the expression levels of Notch1， NICD， and Hes1 proteins in the cells in DMSO+Jagged1 group were increased （P<0.05）； compared with DMSO+sh-PAX3 group， the expression levels of Notch1， NICD， and Hes1 proteins in the cells in DMSO+sh-PAX3+Jagged1 group were increased （P<0.05）.The immunofluorescence results showed that after lentivirus infection， the positive expression of cTnI protein was found in the cells in various groups. Compared with DMSO group and DMSO+sh-NC group， the positive expression of cTnI protein in the cells in DMSO+sh-PAX3 group was decreased， and the differentiation rate of the cardiomyocyte-like cells was decreased （P<0.05）； compared with DMSO group and DMSO+sh-NC group， the positive expression of cTnI protein in the cells in DMSO+Jagged1 group was increased，and the differentiation rate of the cardiomyocyte-like cells was increased （P<0.05）； compared with DMSO+sh-PAX3 group， the positive expression of cTnI protein in the cells in DMSO+sh-PAX3+Jagged1 group was increased，and the differentiation rate of the cardiomyocyte-like cells was increased （P<0.05）. Conclusion PAX3 gene silencing may inhibit the differentiation of the P19 cells into the cardiomyocyte-like，and its mechanism may be realized by decreasing the PAX3 gene expression and regulating the Notch signaling pathway.

Key words: P19 cells, Paired box transcription factor 3, Notch signaling pathway, Cardiomyocyte-like cells