Functional Investigations Of MIR148A Family In Regulating Cardiomyocyte Differentiation Of Human Pluripotent Stem Cells

Human embryonic stem cells(hESCs)can be differentiated into cardiomyocytes in vitro,and thus held great promise in the study of heart development and diseases,cell therapy,cardiac drug screening and toxicity analysis.However,clinical studies and applications of hESC-derived cardiomyocytes are hampered by the shortages of present protocols for cardiac differentiation,which include low efficiency and poor stability of current methods,as well as heterogeneity of the differentiated cardiomyocytes.MicroRNAs(miRNAs)is a kind of non-coding RNAs,about 22 nt in length,which can specifically bind to and degrade the target mRNAs directly,or inhibit their protein translation.MiRNAs have been considered to play critical roles on heart development and cardiomyocyte differentiation.In this study,we observed continuously increased expression of all three members of the MIR148A family,including MIR148A,MIR148B and MIR152,in hESC-derived cells during cardiomyocyte differentiation.To exprelore the roles of MIR148A family,we generated hESC lines with either individual deletion or triple-knockout of MIR 148A family members(MIR148A-TKO),by using CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)-mediated genome-editing.Compared to wild-type hESCs,MIR148A-TKO showed no significant difference in efficiency of cardiac differentiation.Real-time quantitative PCR results indicated a dosage compensation effect of the residual members in these hESCs and their derivates.While the self-renewal and pluripotent capability was not obviously changed between the wild-type hESCs and MIR148A-TKO,the MIR148A-TKO showed significantly reduced cardiac differentiation efficiency,which could be restored by the ectopic expression of MIR148A family members such as MIR152.In addition,MIR152 could also improve the efficiency of cardiac differentiation in wild-type hESCs.By using real-time quantitative PCR method,we found that triple-knockout of MIR 148 A family members in hESCs did not affect the marker gene expression of primitive streak(T,MIXL1),however,significantly inhibited the expression levels of lateral mesoderm(MESP1,FOXF1)and cardiac progenitor(ISL1,NKX2,5)marker genes.RNA-Seq analysis identified a total of 1837 differentially expressed genes in MIR148A-TKO and wild-type hESC-derived cells on day 4 of cardiac differentiaton.Gene Ontology enrichment analysis showed knockout of all MIR148 A family members inhibited lateral mesodermal and cardiac differentiation,while promoted formation of paraxial mesoderm and somites.Interestingly,triple-knockout of MIR148A family members during cardiac differentiation enforced the activation of NOTCH signal pathway.DLL1,a ligand of NOTCH signal pathway,was verified to be a target gene of MIR148A family and up-regulated in MIR148A-TKO on day 4 of cardiac differentiation.Furthermore,knockdown of DLL1 could inhibit downsteam gene expression of NOTCH signal pathway,and increase the cardiac differentiation efficiency of MIR148A-TKO hESCs.Conclusion:The expression levels of all MIR148A family members were progressively increased during the hESC differentiation into cardiomyocytes.MIR 148A family could promote hESC differentiation into cardiomyocytes at least partially by inhibiting DLL1 expression and DLL1-mediated NOTCH signaling pathway.Triple-knockout of MIR148A family members leads to aberrant activation of DLL 1-mediated NOTCH signaling pathway,which promotes paraxial mesodermal/somitic differentiation and inhibits lateral mesodermal/cardiac differentiation.Our findings provide a new insight into heart development and cardiac differentiation.