LncRNA AC125982.2 regulates apoptosis of cardiomyocytes through mir-450b-3p/ATG4B axis in a rat model with myocardial infarction

Fig. 1. Pathological characteristics of rats with myocardial infarction (MI). Rats were subjected to sham operation (C) or myocardial infarction (MI) for four weeks. (A) M-mode ultrasound image of the heart in sham and MI rats. (B) Masson-trichrome staining and heart infarct area (%) in the hearts of rats in sham and MI groups. (C) Left ventricular ejection fractions (LVEF, %) in the hearts of rats in sham and MI groups. (D) Left ventricular shortening fraction (LVFS, %) in the hearts of rats in sham and MI groups. **p < 0.001, n = 3/group.

Fig. 2. Screening and clustering of differentially expressed LncRNAs in MI-rat heart. After the rat model establishment, heart tissues were collected, followed by lncRNA sequencing and qPCR validation. Heat map (A) and scatter plot (B) of differential lncRNA expression in Sham and MI groups. Fold change >1.5, p < 0.05. (C) Upregulation of top 5 LncRNAs in differentially expressed LncRNAs verified by qPCR in heart tissues of rats in Sham and MI groups. *p < 0.05, **p < 0.01, n = 3/group.

Fig. 3. Validation of differentially expressed LncRNAs in Hypoxia-primary cardiomyocytes. Rat primary cardiomyocytes were treated under a hypoxic environment in vitro for different times, mimicking hypoxia after MI in vivo. (A) TUNEL staining to detect apoptosis in primary cardiomyocytes under hypoxia. (B) Counting of the number of apoptotic cells in (A). Versus Control group, **p < 0.01, n = 3/group. (C) The expression of the top five up-regulatedLncRNAs was verified by qPCR in cardiomyocytes subjected to 36 h of hypoxia. *p < 0.05, **p < 0.01, n = 3/group.

Fig. 4. Prediction and validation of miRNAs that bind to AC125982.2. (A) qPCR to verify the binding of 6 miRNAs to AC125982.2 in cardiomyocytes subjected to 36 h of hypoxia. *p < 0.05, **p < 0.01, n = 3/group. (B) qPCR to verify the interference effect of three small interfering RNAs (siRNAs) on AC125982.2 after transfection for 24 h. Versus siNC, **p < 0.01, n = 3/group. (C) qPCR to detect the expression of 6 miRNAs after transfection with siRNA1-LncRNA AC125982.2 for 24 h. *p < 0.05, **p < 0.01, n = 3/group. (D) Downregulation of mir-450b-3p was verified by qPCR in heart tissues of rats in Sham and MI groups. *p < 0.05, n = 3/group. (E) Dual-luciferase assay confirms the target relationship between AC125982.2 and mir-450b-3p. **p < 0.01, ns represents no significant difference, n = 3/group.

Fig. 5. Effect of interfering with AC125982.2 expression on ATG4 and cell apoptosis. (A) The ATG4B and ATG4D protein levels were detected by Western blotting in primary cardiomyocytes treated with hypoxia for 36 h after 24 h of interference with AC125982.2. *p < 0.05; **p < 0.01, n = 3/group. (B) Upregulation of ATG4B was verified by qPCR in heart tissues of rats in Sham and MI groups. **p < 0.01, n = 3/group. (C) Dual-luciferase assay confirms the target relationship between ATG4B and mir-450b-3p. **p < 0.01, ns represents no significant difference, n = 3/group. (D) Western blot detection of cleaved caspase3, Bax and Bcl-2 protein levels in primary cardiomyocytes treated with hypoxia for 36 h after 24 h of interference with AC125982.2. *p < 0.05, **p < 0.01, n = 3/group. (E) qPCR to detect the expression of AC125982.2 and mir-450b-3p after simultaneous interference of AC125982.2 and mir-450b-3p for 24 h in hypoxia-primary cardiomyocytes. *p < 0.05, **p < 0.01, n = 3/group. (F) Western blot assay for simultaneous interference of AC125982.2 and mir-450b-3p for 24 h on ATG4 protein in hypoxia-primary cardiomyocytes. **p < 0.01, n = 3/group. (G) TUNEL staining for simultaneous interference…

Fig. 6. Effect of interfering with ATG4B expression on cell apoptosis. Western blot assay for the ATG4B, Bax, cleaved caspase3 and Bcl-2 protein levels in primary cardiomyocytes treated with hypoxia for 36 h after 24 h of interference with ATG4B. **p < 0.01, ns represents no significant difference, n = 3/group.