miR-301a-PTEN-AKT Signaling Induces Cardiomyocyte Proliferation and Promotes Cardiac Repair Post-MI

Figures

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  Figure 1. Enrichment of miR-301a in the Mammalian Neonatal Cardiomyocytes (A) miRNA screening identified a subset of miRNAs with higher expression in the hearts from neonatal mice than adults. (B) miRNA screening identified a subset of miRNAs with higher expression in the hearts from neonatal rats than adults. (C and D) Validation of miR-301a enrichment in the neonatal cardiomyocytes of mice (C) and rats (D) by qRT-PCR analysis. (E) miR-301a showed more enrichment in primary cardiomyocytes compared to cardiac fibroblasts in neonatal rats. Data are mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01.

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  Figure 2. miR-301a Promoted Cardiomyocyte Proliferation (A) Overexpression of miR-301a promoted cell proliferation in H9C2 cells. (B) The cell cycle analysis indicated an increased G1/S transition in H9C2 cells by miR-301a overexpression. (C) Quantitative analysis of the cells in the S phase before (0 h) and after (12 h) serum release of the cells with or without overexpression of miR-301a. (D) Ki67 and α-actinin co-staining on H9C2 cells with or without overexpression of miR-301a indicating increased double-positive cell percentage by miR-301a. (E) Quantitative analysis of Figure D. (F) Aurora B and α-actinin co-staining on the primary cardiomyocytes from neonatal rat with or without overexpression of miR-301a indicating increased proliferating cardiomyocytes by miR-301a. (G) Quantitative analysis of Figure F. (H) EdU and α-actinin co-staining on the primary cardiomyocytes further demonstrated the cell proliferation induction by miR-301a. (I) Quantitative analysis of Figure H. Data are mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01.

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  Figure 3. miR-301a Protected Cardiomyocytes against Hypoxia-Induced Cell Apoptosis (A and B) H9C2 cells were transfected with RNA oligonucleotide negative control (NC) (A) or miR-301a mimic (B) and cultured under the hypoxia condition with 1% oxygen for 24 h followed by annexin V analysis. The percentages of living cells, early apoptotic cells, late apoptotic cells, and dead cells are indicated. (C) Quantitative analysis indicated fewer apoptotic cells in the miR-301a group compared to the NC. (D) Quantitative analysis indicated more survived cells in the miR-301a group compared to the NC. Data are mean ± SEM (n = 3). ∗p < 0.05.

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  Figure 4. AAV9-Mediated miR-301a Delivery Promoted Post-MI Repair In Vivo (A) Schematic representation of the procedure for tail vein injection of AAV9-miR-301a to the MI model of mice, followed by echocardiography monitoring. (B) AAV9-GFP was applied to the mice by tail vein injection following the exact same protocol as for AAV9-miR-301a administration. GFP signaling and α-actinin staining on the heart tissue in 2 weeks indicating the GFP delivery to cardiomyocytes in vivo. The α-actinin+ cells and GFP+/α-actinin+ cells were counted under a microscope to quantify the delivery efficiency. Data are mean ± SEM (n = 3). ∗∗p < 0.01. (C) Representative echocardiography images of normal control mice, as well as MI mice treated with miR-301a or NC. (D) The ejection fraction change of the individual mice in the control group (n = 10) from day 0 before surgery, day 3 before miRNA injection, day 15 after miRNA injection, and day 30 before animal sacrifice. (E) The ejection fraction change of the individual mice in miR-301a group (n = 10). (F) The average ejection fraction values at the above time points of the mice in the PBS control group, the miR-301a-treated MI group, and the NC-treated MI…

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  Figure 5. PTEN is a Target Gene of miR-301a in Cardiomyocytes (A) Comparison between 2,209 predicted target genes of miR-301a in rats, 2,345 predicted target genes of miR-301a in mice, and 487 genes related to heart development. (B) The list of 21 genes overlapped in Figure A. (C) qRT-PCR analysis validated downregulation of PTEN, SMAD4, TIMP2, and TGFBR2 expression by miR-301a in cardiomyocytes. (D) Sequence BLASTN analysis identified three binding sites to miR-301a at the 3′ UTR region of PTEN mRNA. The three binding sites are highly conserved between humans, mice, and rats. Luciferase reporter vectors carrying either wild-type (WT) or point-mutated (MU, mutated nucleotides are indicated with red font) PTEN 3′ UTR were cloned. (E) Luciferase reporter assays demonstrated the increased luciferase activity in the MU vector compared to the WT. Co-transfection of miR-301a inhibited luciferase activity in the WT, but not MU, vector. Data are mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01.

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  Figure 6. PTEN/PI3K/AKT Signaling Mediated miR-301a Regulation of Cell Proliferation in Cardiomyocytes (A) miR-301a mimic, PTEN siRNA, and an NC were applied to H9C2 cells, respectively, followed by EdU staining, indicating that both PTEN knockdown and miR-301a overexpression promoted cardiomyocyte proliferation. (B) Quantitative analysis of (A). (C) Western blot analysis demonstrating the upregulation of PTEN at the protein level in H9C2 cells after transfection with PTEN plasmid. (D) Introduction of PTEN back into the miR-301a-treated H9C2 cells attenuated the cell proliferative induction by miR-301a. (E) Western blot analyses demonstrating that upregulation of p-AKT and p-GSK-3β were associated with downregulation of PTEN in the hearts from AAV9-miR-301a-treated mice. (F) Western blot analyses demonstrating increased p-AKT and decreased p-GSK-3β and cyclin D1 in H9C2 cells by application of RG7440, a small molecule inhibitor of AKT. (G) CCK-8 assays demonstrating that RG7440 treatment in H9C2 cells not only suppressed cell proliferation via inhibiting endogenous AKT activity (Ctrl groups), but also reversed the miR-301a-induced cell proliferation (miR-301a groups). (H) Cartoon…