| The differentiation of mesodermal cells is notably complicated,they can differentiate and develop into various organs and tissues.The heart,as the vital engine of life,is the first developed fetal organ.Red blood cells are the carrier of O2 and CO2.Both of them develop from mesoderm,playing very important roles in life.Abnormalities in the heart and blood system can induce a plurality of diseases.In addition,there is a close correlation between these diseases:hemoglobin related abnormalities are often accompanied by cardiovascular diseases,such as left ventricular dysfunction which includes myocarditis,myocardial ischemia,and even heart failure,which could cause death.Researches in directed cell differentiation bring hope for the treatment of related diseases.Therefore,we studied the process of cell differentiation in mesoderm,in order to find new functional genes.By screening with random siRNA library our lab found that Hmgn5 and long non-coding RNA hLOC correlated with cardiac and erythroid differentiation,respectively.Up to date,there is neither report about the function of Hmgn5 in early embryo differentiation and myocardial differentiation,nor any report of LncRNA hLOC function.We set up two cell differentiation models:cell aggregation induction of ES and p19 cells for myocardial differentiation;and anti-tumor drug Ara-C induction of K562 cells for erythroid differentiation.The studies of Hmgn5 and hLOC were carried out on these models.We found Hmgn5 played roles in myocardial differentiation from mesodermal cells.Its time-specific expression pattern(increased transiently and then decreased)is specific in the process of myocardial differentiation.Down-regulation of Hmgn5 delayed the increased expression of GATA-4 and inhibited the expression of Nkx2.5;and inhibited the expression of pacemaker makers:Hcn4 and Tbx3.By overexpressing Hmgn5 we proved that Hmgn5 promoted the expression of Tbx3,Hcn4;inhibited the expression of working cardiomyocytes markers:a-MHC,My17 and ?-actin;and decreased the area of beating myocardial cells.Knocking down of GATA-4 or Nkx2.5 downregulated the expression of Hmgn5.Moreover,both ChIP and dual-luciferase reporter assay confirmed that GATA-4 and Nkx2.5 bound to Hmgn5 promoter and activated it,which showed that GATA-4 and Nkx2.5 could promote the expression of Hmgn5.The above results demonstrated that,GATA-4 and Nkx2.5 activated Hmgn5 expression by directly binding to its promoter;there is a mutual regulatory relationship between GATA-4,Nkx2.5 and Hmgn5.Then Hmgn5 regulated pacemaker and working cardiomyocytes related genes,indicating that Hmgn5 have different functions and mechanisms in the differentiation of different types of cardiomyocytes.Another novel functional gene we found——long noncoding RNA hLOC involved in the regulation of mesoderm-derived erythroid differentiation.hLOC has two splicing variants:hLOC and hLOC50.The RNA level of the two variants significantly increased during erythroid differentiation.We did find that IGF2BP1 mRNA level was significantly down-regulated during this process.We also found that hLOC knockout promoted the expression of y-globin and inhibited the expression of ?-globin.And interestingly,IGF2BP1 mRNA level was elevated.IGF2BP1 is known to shift HbA to HbF by inducing the expression of y-globin and inhibiting ?-globin.So,LncRNA hLOC inhibited IGF2BP1 and y-globin expression and promoted ?-globin expression.In summary,Hmgn5 and LncRNA hLOC were identified as new functional genes involved in the regulation of mesoderm-derived cardiomyocyte differentiation and erythrocyte differentiation,respectively.Hmgn5 is activated directly by GATA-4 and Nkx2.5,having different functions and mechanisms in the differentiation of working cardiomyocytes and pacemaker cells.And for hLOC,it could inhibit IGF2BP1 and y-globin expression and promote ?-globin expression... |
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