| Smooth muscle is composed of smooth muscle cells responsible for vascular contraction and extracellular matrix generation.Phenotypic transformation of smooth muscle cells is associated with various cardiovascular diseases,including atherosclerosis and hypertension.The Pbx1 gene is a TALE homeodomain transcription factor that encodes a transcription factor of the PBX homeodomain family and plays an important role in embryonic development and organogenesis.In this study,we utilized an established embryonic stem cell(ESC)directed differentiation into smooth muscle cells(ESC-SMC)in vitro differentiation model,combined with RNA-Seq data analysis,to reveal that Pbx1 may be involved in the differentiation process of smooth muscle cells.Homozygous knockout of the Pbx1 gene in ESC cell lines further indicated that Pbx1 knockout inhibited smooth muscle cell differentiation.We constructed ESC cell lines overexpressing Pbx1 using lentiviral transduction,and used the ESC-SMC differentiation model for phenotype observation and detection of SMC marker gene expression.Our results confirmed that Pbx1 overexpression promotes ESC differentiation into SMCs.To explore the molecular mechanism involved,we utilized RNA-Seq data,the JASPAR database,literature analysis,and q PCR verification to identify several candidate target genes of Pbx1.Subsequently,we designed dual-luciferase experiments and point mutation experiments to verify its mode of action.Existing data suggests that Pbx1 gene promotes SMC differentiation,but is not directly targeted to the Epas1 gene.In addition,we also verified the function of Pbx1 using another SMC in vitro differentiation model(10T1/2 cells),which showed that overexpression of Pbx1 is sufficient to promote 10T1/2 cell differentiation into smooth muscle-like cells.MTT experiments also showed that Pbx1 overexpression promotes 10T1/2 cell proliferation.In conclusion,our study demonstrates that Pbx1 plays an important role in the process of SMC differentiation,providing insights into the mechanism of smooth muscle differentiation and potential drug development and clinical treatment for cardiovascular diseases. |
