MicroRNA-1 Regulates Bone Marrow Mesenchymal Stem Cells Differentiation Into Cardiomyocyte-like Cells

Background and Objective Mesenchymal stem cells from bone marrow are multipotent stem cells that can differentiate into multiple cell types. It has been shown that MSCs can differentiate into cardiomyocytes, transplantation MSCs in damaged heart, the heart function improved. However, it has also been demonstrated that the efficiency of MSCs differentiated into cardiomyocytes in vivo is low and the effect of repairing damaged heart is scant. Therefore, promoting the differentiation of MSCs into myocardial cells and improving the efficiency are the urgent issues need to address for optimal treatment of ischemic heart disease. miRNAs are genomically encoded highly conserved, noncoding approximately 18-22 nt RNAs that negatively regulate gene expression by either promoting the degradation of mRNA or down-regulating the protein production by translational repression. Recent findings revealed a muscle-specific miRNA-1 plays important roles in heart development and muscle differentiation. These studies suggested that miRNA-1 can promote embryonic stem cells and cardiac progenitor cells differentiated into cardiomyocytes. It was reported that transfection of miRNA-1 in HeLa and C2C12 cells has been shown to shift the gene expression profile toward that of muscle cells. But the roles of miRNA-1 in MSCs differentiated into CMs remain to be clarified. In this study, an eukaryotic expression vector for rno-miRNA-1 was constructed successfully and MSCs were transfected with it. We have analyzed the expression of cardiac-related genes GATA4, NKx2.5, MEF2C, cTnI and explored the effects of miRNA-1 on MSCs differentiated into cardiomyocyte-like cells. The results demonstrated that miRNA-1 can induce MSCs differentiated into cardiomyocyte-like cells. miRNA-1 may enhance the efficiency of MSCs differentiated into cardiomyocytes and be a new target for the therapies of ischemic heart disease with MSCs.Methods miRNA-1 genomic sequence was amplified by PCR and cloned into pSD11-U6/Neo/GFP plasmid, rno-miRNA-1 eukaryotic expression vector was constructed, rat MSCs were isolated, amplified and identified. The fourth passage of MSCs were transfected miRNA-1 plasmid with Lipofectamine2000. miRNA-1 expression was detected by qRT-PCR, the mRNA expression of cardiac-specific transcription factor GATA4, NKx2.5 and MEF2C were detected by RT-PCR,cTnI expression was measured by immunofluorescence,respectively,2, 4,6 days.Results PCR identification and DNA sequencing showed that the eukaryotic expression vector for miRNA-1 constructed successfully. MSCs showed a fibroblast-like morphology and whirlpool-like distribution. Immunofluorescence showed that more than 90% of the MSCs were positive for integrin family member CD29, adhesion molecules CD44,which are two significant markers of MSCs and negative for hematopoetic precursor cell markers CD34,leukocyte antigen CD45. Transfection of miRNA-1 in MSCs 48h later, 20-30% of MSCs expressed GFP and qRT-PCR analysis showed that the expression of miRNA-1 was markedly enhanced. The results of RT-PCR showed that the expression of GATA4, NKx2.5 and MEF2C gradually increased. The results of immunofluorescence showed that the cTnI expression was detected by day 4,6. In untransfected group and transfected empty plasmid group, the expression of GATA4, NKx2.5 and cTnI were not examined and the expression of MEF2C had no change.Conclusions The eukaryotic expression vector for miRNA-1 was constructed succes- sfully. It may enhance the expression of miRNA-1 in MSCs effectively. miRNA-1 has the important role that induces MSCs differentiated into cardiomyocyte-like cells and be a new target for the therapies of ischemic heart disease with MSCs. .