Abstract 3225: Inhibition of microRNAas762, 762+ 21, and microRNAs762+31, Differentiates Cardiac Stem Cells into Cardiomyocytes by Activation of TGFβIII and Activin A Receptors Pathways
We hypothesized that inhibition of specific miRNA in mouse cardiac stem cells (CSC) and CSC with GATA-4 (CSCG) could induce differentiation into cardiomyocytes. To test this hypothesis we first studied the miRs that were predominant in mouse cardiac stem cells (CSC) and in cardiac stem cells with GATA4 inserted (CSCG), but absent in adult mouse hearts(MHtC). Then we inhibited those miRNAs with specific antisense. Cells to changed to beating cells indicating differentiation. To test the mechanism of this differentiation we tested for activation target genes those miRNAs. We studied 569 unique miRNAs probes in MHtC, CSC and CSCG. We identified uniquely high expression of miR762, miR21 and miR 31 in CSC and CSCG compared to MHtC. CSC and CSCG were cultured in matrigel and anti-mRNAs anti-sense to miR-762; 762+21; 762+31and 762+21+31 was tested by 1, 5, 10, 50 and 100nM with oligo-fectamine. Beating cells were recorded on confocal microscopy. The target genes of these miRs: TGF-βIII, activin A (receptors type-IB, II-like-1, 2a) BMP (receptors type 2),and transcription factors Smad-4 and Dicer were analyzed by RT-PCR, Also cardiac sarcomeric proteins (α and β-MHC, MLC-2a, MLC-2v and cTNI) were tested.. The maximum miRNA expression inhibition was observed 6hs after the treatment As a positive control the cells were treated with activin A and BMP-4 cytokines and also compared to MHtC expression. In CSCs, antisense inhibition of miR762 + 21 was the most effecitive in inducing beating cells. The miR-762, 762+21 and 762+21+31 anti-sense activated the target genes TGFβIII, AcvR (AcvR 2a and like-1), but not BMPR. Transcription factor Smad-4, MLC-2a, and α-MHC expression increased with miR762+21 anti-sense. In CSCG cells, miR762+31 and 762+21+31 inhibition increased expression of their target genes, including TGFβIII, AcvR (2a and IB), but not BMPR. Also, Smad4 and MLC-2a and α-MHC were activated. We conclude that miR762+miR 21 in CSC and miR-762+31in CSCG, hold back cell differentiation by inhibiting their target genes. When these miRs are inhibited specifically stem cells differentiate into beating cells with cardiomyocyte markers.