LncRNA CACF promotes autophagy and cardiac recovery after myocardial infarction by targeting ATG7

Figures

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  Figure 1. Identification and functional characterization of differentially expressed lncRNAs in infarcted cardiac tissues (A) Expressions of lncRNAs in MI tissues, compared to normal tissues in pigs. (B) Expression profiles of five lncRNAs across different tissues. (C) Expressions of five lncRNAs in the infarcted myocardium and normal myocardium. (D) Expressions of five lncRNAs in the human genome. (E) Changes in the expressions of five lncRNAs in vascular cells treated with their overexpression plasmids and smart silencers, respectively. (F) Influences of five lncRNAs on the number of tubes formed by vascular cells. Effects of five lncRNAs on the mRNA (G) and protein (H) levels of their target genes. Data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01; unpaired two-tailed Student’s t test. Scale bars, 200 μm in (F); n = 3 biological replicates per group.

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  Figure 2. CACF promoted proliferation and cell cycle progression and inhibited the apoptosis of vascular cells (A) Effect of CACF on the cellular proliferation. Effects of pcDNA3.1-CACF (B) and CACF silencer (C) on the viabilities of vascular cells. Effects of CACF on the mRNA (D) and protein (E) levels of proliferation-related genes. Effects of CACF on the cell cycle (F) and apoptosis (G). Effects of CACF on the levels of cell cycle-related genes (H), apoptosis-related genes (I), and apoptosis-related proteins (J). Data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01; unpaired two-tailed Student’s t test. Scale bars, 100 μm in (A); n = 3 biological replicates per group.

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  Figure 3. CACF might play a crucial role in MI by promoting autophagy (A) The electrocardiogram of rats after LAD. (B) Changes of histological and pathological of rat heart tissues after MI. (C) The expression of CACF in heart after MI of rats. (D) The expressions of CACF in the genomes of humans, rats, and pigs. (E) The distribution of CACF in the cytoplasm and nucleus in vascular cells. U1 small nuclear RNA was used as an endogenous nuclear reference, whereas GAPDH was used as a cytoplasmic reference. (F) Effect of CACF on the formation of autophagy flux in vascular cells. Effect of CACF on the levels of autophagy-related genes (G) and autophagy-related proteins (H). Data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01; unpaired two-tailed Student’s t test. Scale bars: 25 μm in (B) and 10 μm in (F); n = 3 biological replicates per group.

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  Figure 4. CACF promoted autophagy by competitively binding to miR-520b-3p and miR-20a-5p to promote the expression of ATG7 (A) The mRNA level of ATG7 in vascular cells treated with pcDNA3.1, pcDNA3.1-ATG7, si-NC, and si-ATG7. (B) Effect of ATG7 on the proliferation of vascular cells. (C) Effect of pcDNA3.1-ATG7 on the viability of vascular cells. (D) Effect of si-ATG7 on the viability of vascular cells. Effect of ATG7 on the cell cycle (E) and apoptosis (F). (G) The levels of miR-520b-3p and miR-20a-5p in CACF-sense and CACF-antisense pull-down complexes. (H) The miRNA levels of miR-520b-3p and miR-20a-5p in vascular cells treated with mimics and silencers, separately. (I) The fluorescence activity of CACF after co-transfection of wild-type/mutant CACF, miR-520b-3p mimic/mimic NC and miR-20a-5p mimic/mimic NC. (J) Effect of CACF on the miRNA levels of miR-520b-3p and miR-20a-5p in vascular cells. (K) Effects of miR-520b-3p and miR-20a-5p on the level of CACF in vascular cells. (L) The fluorescence activity of ATG7 after co-transfection of the wild-type/mutant ATG7 3′ UTR, miR-520b-3p mimic/mimic NC and miR-20a-5p mimic/mimic NC. (M) Effects of miR-520b-3p and miR-20a-5p on the level of…

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  Figure 5. MiR-520b-3p inhibited proliferation and the cell cycle and promoted the apoptosis of vascular cells, while miR-20a-5p had the opposite effect Effects of miR-520b-3p (A) and miR-20a-5p (B) on the proliferation of vascular cells. Effects of miR-520b-3p mimic (C) and inhibitor (D) on the viability of vascular cells. Effects of miR-20b-5p mimic (E) and inhibitor (F) on the viability of vascular cells. Effects of miR-520b-3p (G) and miR-20a-5p (H) on the progression of cell cycle of vascular cells. Effects of miR-520b-3p (I) and miR-20a-5p (J) on the apoptosis of vascular cells. Effect of miR-520b-3p on the expression levels of proliferation marker genes and cell cycle marker genes (K). Effect of miR-20a-5p on the expression levels of proliferation marker genes and cell cycle marker genes (L). Effect of miR-520b-3p (M) and miR-20a-5p (N) on the expression levels of apoptosis marker genes. Effect of miR-520b-3p on the levels of proliferation marker proteins (O) and apoptosis marker proteins (P), and the detected proteins were derived from the same membrane or obtained through stripping and reprobing. Effect of miR-20a-5p on the levels of proliferation marker proteins (Q) and apoptosis…

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  Figure 6. MiR-520b-3p and miR-20a-5p inhibited autophagy in vascular cells (A) Effects of miR-520b-3p mimic and miR-20a-5p mimic on the autophagy in vascular cells. (B) Effects of miR-520b-3p inhibitor and miR-20a-5p inhibitor on autophagy in vascular cells (scale bars, 100 μm). Effects of miR-520b-3p (C) and miR-20a-5p (D) on the expression levels of autophagy marker genes in vascular cells. Effects of miR-520b-3p (E) and miR-20a-5p (F) on the expression levels of autophagy marker proteins in vascular cells. (G) Expressions of miR-520b-3p and miR-20a-5p in the hypoxic environment of MI simulated by H2O2. Data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01; unpaired two-tailed Student’s t test. Scale bars, 100 μm in (A and B); n = 3 biological replicates per group.

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  Figure 7. CACF regulated autophagy to promote cardiac recovery after MI (A) The level of CACF in rat hearts with MI after the injection of AAV9-CACF/NC and AAV9-shRNA/NC. The levels of miR-520b-3p and miR-20a-5p in the hearts of MI rats (B), and the level of ATG7 in the hearts of MI rats (C). The levels of miR-520b-3p and miR-20a-5p in the hearts of MI rats (D), and the level of ATG7 in the hearts of MI rats (E) after the injection of AAV-9-CACF/NC or AAV9-shRNA/NC. (F) The survival rate of rats with MI in each treatment group. (G) Changes of histological and pathological in the hearts of rats with MI after the injection of AAV-9-CACF/NC or AAV9-shRNA/NC. (H) The protein levels of ATG7, ATG5, p62, and CD31 in the hearts of rats with MI after the injection of AAV-9-CACF/NC or AAV9-shRNA/NC. Effect of hypoxic conditions on proliferation (I), viability (J), cell cycle (K), and apoptosis (L) of vascular cells. (M) Effect of hypoxic conditions on autophagy of vascular cells. (N) The expressions of autophagy marker genes in vascular cells during hypoxic conditions. (O) The levels of CACF and ATG7 in vascular cells during hypoxic conditions. Data are shown as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01;…