The Function And Mechanism Of LncCMRR In Cardiomyocyte Differentiation Of Embryonic Stem Cells

During the early embryonic development of vertebrates,the heart is the first organ formed and regulated by a series of transcription factors,epigenetic regulatory factors and signaling pathways.The disorder of heart development regulatory networks can lead to congenital heart diseases,teratogenesis,and embryonic death.It has been reported that the origins of some heart diseases in adolescents and adults can also be traced to the early stages of heart development.However,the understanding of the pathogenesis of heart diseases is still limited.Therefore,exploring the specific regulatory mechanisms in heart development will play a critical role in the understanding,diagnosis,prevention and treatment of heart diseases.Long noncoding RNAs(LncRNAs)are defined as a class of noncoding RNA transcripts of more than 200 nucleotides with low-coding potential for protein.As critical epigenetic regulators,lncRNAs play an important role in the growth,development,behavior,cognition and disease of organisms.In recent years,lncRNAs have been paid more attention to heart development and heart diseases.However,due to the complexity of heart development and the diversity of regulatory mechanisms of lncRNAs,the exact regulatory mechanisms of specific lncRNAs in heart development and heart diseases still need to be further studied.In this study,we identified one lncRNA as playing a crucial role in cardiac mesoderm differentiation,and named it as cardiac mesoderm-related lncRNA(LncCMRR),which was up-regulated in the embryoid body differentiation of mouse embryonic stem cells(ESCs)and the development process of mouse embryos.Knockout(KO)of LncCMRR in ESCs did not affect the self-renewal of ESCs,but damaged the formation of cardiac mesoderm and affected the efficiency of cardiomyocyte differentiation.In vivo experiments in mice showed that the expression level of cardiac mesoderm marker such as Flk1 was significantly decreased in E7.75 of LncCMRR-KO mice.In addition,the thickness of the myocardium and cell number of mouse heart at E17.5 of LncCMRR-KO mice was significantly decreased.Furthermore,the heart function of LncCMRR-KO mice was significantly decreased at adulthood,accompanied by cardiac hypertrophy and cardiac dilation.These results suggested that the deletion of LncCMRR is very likely to damage the normal development of mouse heart,resulting in the occurrence of cardiac dysfunction in mice.Further mechanistic analyses showed that a conserved purine-rich negative regulatory(PNR)element in mammals located at the promoter region of the key gene Flk1 in cardiac mesoderm.And the transcriptional suppressor protein PURB could specifically identify and bind to the PNR element in the gene promoter region to inhibit the expression of downstream related genes.This study found that LncCMRR mainly acted as a molecular bait by directly binding to PURB,preventing PURB from enriching on the PNR element in the Flk1 promoter region,thereby protecting the normal transcription of Flk1,and ultimately ensuring the normal progress of cardiac mesoderm,as well as cardiomyocyte differentiation process.In summary,we have found a new lncRNA(LncCMRR)that regulates cardiac mesoderm differentiation,and elucidated the function and regulatory mechanism of LncCMRR in cardiomyocyte differentiation of mouse ESCs and cardiac development.These findings not only enrich the epigenetic regulatory network of lncRNAs to regulate cardiomyocyte differentiation,but also develop the understanding of lncRNAs in mediating the defects of heart development.