MiR-1Mediating Of Notch Signaling Promotes Rat Mesenchymal Stem Cells Differentiation Into Cardiac-Like Cells

ObjectiveTransplantation of mesenchymal stem cells （MSCs） has emerged as a potentialtreatment for ischemic heart repair. Studies have suggested that muscle specificmiR-1can function as a key regulator in cell differentiation. Notch signaling pathwayis one of the most important pathway during development and play key roles in cellproliferation, apoptosis, and differentiation. It has also been reported that there mightbe an interaction between Notch signaling and miRNAs, through which miRNAsregulates Notch signaling pathway and exerts its function. Therefore, wehypothesized that miR-1induction into rat MSCs can induce MSCs differentiationinto cardiac phenotypes through regulating Notch signaling pathway. This projectlays foundation for cell-based therapies of ischemic heart diseases.MethodsMSCs were isolated from rat bone marrow by the whole bone marrow adherencemethod; cell morphology were detected through inverted light microscope; MSCsidentified by flow cytometry were transduced with both miR-1and green fluorescentprotein （GFP）(MSCs^miR-1) using the murine lentiviral expression system, controlcells were only GFP transduced. After incubated with miR-1lentiviral expressionsystem for15days, the cell morphology were examied by light microscope; On day0,4,6,15after transduction, MSCs^miR-1cells were detected for the expression ofmiR-1, cardiac-specific genes GATA-4, Connexin43（Cx43）, cTnI and α-actin, as wellas Notch signaling-related genes by real-time quantitative polymerase chain reaction（qRT-PCR）; The protein level of cTnI and Cx43were detected byimmunofluorescence, α-actin were detected by Western blotting.ResultsIsolated MSCs displayed a stable spindle-phenotype and showed characteristicswirling growth. More than98%of the MSCs population express CD44and CD29for MSCs phenotype; Meanwhile, less than1%cells were CD45positive. Compared with control cells, MSCs^miR-1cells highly express miR-1and show a higherexpression of cardiac-specific genes, including GATA-4, Cx43, cTnI and α-actin,cTnI and Cx43were detected by immunofluorescence in MSCs^miR-1cells after miR-1transduction for4days, and reached the maxed on day15. Western blotting furtherconfirmed the expression of α-actin in MSCs^miR-1cells. By checking the expression ofNotch signaling of mRNA potential targets within, miR-1was seen to be a regulatorof the Notch pathway-related genes, we found that miR-1seems enhance MSCsdifferentiation through regulating Notch ligand Jagged1. Down-regulation of Jagged1mRNA expression by miR-1regulates cell differentiation, and induces cardiac-likedifferentiation in rat MSCs. At the same time we also found that the mRNAexpression of Notch1/Notch3and Hey2reduced significantly in MSCs^miR-1cells,which indicates that the down-regulation of Jagged1-Notch1/Notch3-Hey2might beinvolved in miR-1-mediated cardiac-like cell differentiation. This result suggeststhat Jagged1-Notch1/Notch3-Hey2are key regulators in Notch signaling-mediated incardiac-like cell differentiation, and modulation of this pathyway can determine stemcell fate.ConclusionsOur study suggests that transduction of miR-1into rat MSCs induce celldifferentiation into cardiac-like cells by down-regulation mRNA expression ofJagged1-Notch1/Notch3-Hey2in the Notch pathway.