LncRNA SNHG15 promotes angiogenesis and improves cardiac repair after myocardial infarction through MiR-665-mediated KDR expression

Figures

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  Fig. 1. SNHG15 is upregulated in ECs during the post-MI proliferative phase and is expressed in the cytoplasm of HCAECs. (A), Adult mice were subjected to MI or Sham procedure. Hearts were collected 7 days post-MI, n = 4. (B), Cytokine concentrations of VEGF, IL-10, and TGF-β1 in the infarct border and infarct zone were detected by ELISA, n = 6. (C), Hierarchical cluster heat map from lncRNA sequencing data in the peri-infarction 7 days post-MI (n = 4) and selection strategy of SNHG15. (D), SNHG15 level measured by RT-qPCR analysis of cardiac cells isolated from the peri-infarction region of mouse hearts 7 days post-MI or sham surgery, n = 6. Cell isolation was performed using magnetic bead-based cell sorting. (E - F), Time course of the relative SNHG15 level measured by RT-qPCR in cardiac tissues and ECs in the infarct border zone after MI, n = 6. (G), Representative and quantitative analysis of KDR protein levels in ECs of the infarct border zone after MI by western blotting, n = 6. (H), Endogenous SNHG15 (red) in HCAECs detected by RNA FISH. Probes for U6 (red) and 18 S (red) serve as nuclear and cytoplasmic controls, respectively. Scale bar, 20 μm. Data are expressed as mean ± SEM.…

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  Fig. 2. SNHG15 regulates HCAECs proliferation, migration, and tube formation by upregulating KDR. (A), Efficiency of knocking down and overexpressing SNHG15 by RT-qPCR, n = 3. (B), Representative and quantitative KDR protein levels in HCAECs after the knockdown and overexpression of SNHG15 by Western blotting detection, n = 3. (C), Representative and quantification of AKT/eNOS/ERK/Raf1/p38 expressions and phosphorylation in HCAECs treated with the knockdown and overexpression of SNHG15 following western blotting detection, n = 3. (D and E), Cell proliferation was measured through EdU staining and quantification of EdU-positive cells at 6 h after the knockdown and overexpression of SNHG15. Scale bar = 20 μm, n = 3. (F and G), Migration ability was measured by the transwell assay and quantification of transwell cells at 24 h after the knockdown and overexpression of SNHG15. Scale bar = 200 μm, n = 3. (H and I), Migration ability was measured through wound healing assays and migration rate quantification at the indicated time points. Scale bar = 200 μm, n = 3. (J and K), The angiogenic response was measured by conducting tube formation assay and quantification analysis of the total tube…

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  Fig. 3. SNHG15 in ECs promotes therapeutic angiogenesis to against cardiac remodeling and dysfunction post-MI. (A), Schematic diagram depicting the time course of MI-induced cardiac remodeling and dysfunction in mice receiving AAV-SNHG15 or AAV-Scr. (B), Kaplan–Meier survival analysis of mice treated with sham (n = 8), MI (n = 22), MI + AAV- Scr (n = 23) and MI-AAV-SNHG15 (n = 23) post-MI. (C and D), Echocardiography and measured LVEF, FS, LVEDD and LVSDD in mice, n = 6. (E and F), Hematoxylin-eosin staining, Masson’s trichrome, and quantification of cardiac fibrosis, n = 6. (G), Representative and quantitative of KDR protein levels in the ECs of infarct border zone after MI through western blot detection, n = 6. (H), Representative and quantification of AKT/eNOS/ERK/Raf1/p38 expressions and phosphorylation in the border zone were analyzed using western blotting, n = 6. (I and J), Representative images and quantification of immunofluorescence staining for CD31+/Ki67 + cells in the border zone of infarcted hearts at 7 days post-MI. Scale bar = 20 μm, n = 6. (K and L), Representative images and quantification of immunofluorescence staining for CD31 + cells in the border zone of infarcted…

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  Fig. 4. LncRNA SNHG15 targets miR-665 to act as a molecular sponge in HCAECs. (A), The predicted binding sites of SNHG15 and hsa-miR-665 and the mutated binding sites are indicated. (B), Luciferase activities of reporter vectors containing luciferase genes and a fragment of SNHG15 RNA containing wild-type or mutated hsa-miR-665 binding sites, n = 3. (C), SNHG15 was associated with hsa-miR-665. HCAECs were transfected with biotinylated wild-type hsa-miR-665 (Bio-hsa-miR-665-WT) or biotinylated mutant hsa-miR-665 (Bio-hsa-miR-665-Mut), n = 3. (D-E), The RIP assay for SNHG15 was performed with an anti-AGO2 antibody in HCAECs transfected with NC or mimics, and the expression of SNHG15 and hsa-miR-665 was detected, n = 4. Data are expressed as mean ± SEM. Kruskal-Wallis followed by Dunn test (B). One-way ANOVA followed by Dunnett multiple comparisons test (C). Two-way ANOVA followed by Bonferroni multiple comparisons test (D, E). HCAECs, human coronary artery endothelial cells

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  Fig. 5. Hsa-miR-665 negatively regulates HCAECs function and its inhibition restores SNHG15-deficient angiogenesis. (A, D), Cell proliferation was measured by EdU staining and quantification of EdU-positive cells at 6 h after the overexpression and knockdown of hsa-miR-665. Scale bar = 20 μm, n = 3. (B, E), Migration ability was measured by the transwell assay and quantification of transwell cells at 24 h after the overexpression and knockdown of hsa-miR-665. Scale bar = 200 μm, n = 3. (C, F), The angiogenic response was measured using tube formation assay and quantitative analysis of the total tube length and branches. Scale bar = 50 μm, n = 3. The images were obtained from three distinct random microscopic fields within each group. (G, J), Cell proliferation was measured through EdU staining and the quantification of EdU-positive cells at 6 h after the HCAECs are transfected with siRNA for SNHG15 knockdown and subsequently, with the inhibitor of miRNA-665. Scale bar = 20 μm, n = 3. (H, K), Migration ability was measured by the transwell assay and quantification of transwell cells at 24 h after the HCAECs are transfected with siRNA for SNHG15 knockdown and subsequently, with the…

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  Fig. 6. Hsa-miR-665 inhibits HCAECs function by decreasing KDR level. (A), The predicted binding sites of KDR and hsa-miR-665 and the mutated binding sites. (B), Luciferase activities of reporter vectors containing luciferase genes and a fragment of KDR RNA containing wild-type or mutated hsa-miR-665 binding sites, n = 3. (C), KDR was associated with hsa-miR-665. HCAECs were transfected with biotinylated wild-type hsa-miR-665 (Bio-hsa-miR-665-WT) or biotinylated mutant hsa-miR-665 (Bio-hsa-miR-665-Mut), n = 3. (D-E), HCAECs were transfected with concentration gradients of the hsa-miR-665 mimic and inhibitor, and quantitative analysis of the alteration of hsa-miR-665 and the gene and protein expression of KDR was performed, n = 4. Data are expressed as mean ± SEM. Kruskal-Wallis followed by Dunn test (B). One-way ANOVA with Dunnett multiple comparisons test (C-E). HCAECs, human coronary artery endothelial cells; KDR, kinase domain-containing receptor

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  Fig. 7. Overexpression of KDR alleviates SNHG15-deficient HCAECs proliferation, migration, and tube formation. (A, D), Cell proliferation was measured through EdU staining and the quantification of EdU-positive cells at 12 h after the HCAECs are transfected with siRNA for SNHG15 knockdown, followed by overexpression of KDR. Scale bar = 20 μm, n = 3. (B, E), Migration ability was measured by using the transwell assay and quantification of transwell cells at 24 h after the HCAECs are transfected with siRNA for SNHG15 knockdown, followed by overexpression of KDR. Scale bar = 200 μm, n = 3. (C, F), The angiogenic response was measured by tube formation assay and quantitative analysis of the total tube length and branches at 4 h after the HCAECs are transfected with siRNA for SNHG15 knockdown, followed by the overexpression of KDR. Scale bar = 50 μm, n = 3. The images were obtained from three distinct random microscopic fields within each group. Data are the mean ± SEM. Two-way ANOVA followed by Bonferroni multiple comparisons test. HCAECs, human coronary artery endothelial cells; KDR, kinase domain-containing receptor

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  Fig. 8. Overexpressing KDR reversed the exacerbation of MI and EC dysfunction induced by silencing SNHG15 in MI hearts. (A), Schematic diagram depicting the time course of MI-induced cardiac remodeling and dysfunction in mice receiving AAV-shRNA-SNHG15 or AAV-KDR. (B), Kaplan–Meier survival analysis of mice treated with Sham + shRNA- Scr + Scr (n = 8), MI + shRNA- Scr + Scr (n = 20), MI + shRNA-SNHG15 + Scr (n = 27) and MI + shRNA-SNHG15 + KDR (n = 20) post-MI. (C, D), Echocardiography and measured LVEF, FS, LVEDD and LVSDD in mice, n = 6. (E, F), Hematoxylin-eosin staining, Masson’s trichrome, and quantification of cardiac fibrosis, n = 6. (G, H), Representative images and quantification of immunofluorescence staining for CD31+/Ki67 + cells in the border zone of infarcted hearts at 7 days post-MI, n = 6. Scale bar = 20 μm, n = 6. (I, J), Representative images and quantification of immunofluorescence staining for CD31+ cells in the border zone of infarcted hearts at 28 days post-MI. Scale bar = 20 μm, n = 6. Data are the mean ± SEM. HRF, high resolution field. The survival rate was analyzed by Kaplan-Meier method and compared by log-rank test (B). Two-way ANOVA with Bonferroni multiple…

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  Fig. 9. Working model summarizing the function of SNHG15 in VEGF signaling and angiogenesis gene expression. During the proliferative phase of MI, naturally increased expression of SNHG15 in the peri-infarct border ECs, under the stimulus of hypoxia, intercellular communication or pro-angiogenic factors et al., results in the supplement of KDR and enhancing the VEGF signaling. Exogenous massive supplementation of SNHG15 in ECs can greatly improve endogenous KDR ligands without the addition of VEGF proteins to reduce ventricular remodeling and heart failure by increasing angiogenesis and improve heart function after MI. Thus, modulating SNHG15 expression in peri-infarct ECs may be a potential therapeutic strategy to promote cardiac function and remodeling after MI. MI, myocardial infarction; VEGF, vascular endothelial growth factor; KDR, kinase insert domain receptor