MicroRNA-582-5p targeting Creb1 modulates apoptosis in cardiomyocytes hypoxia/reperfusion-induced injury

Abstract

Background: Myocardial ischemia-reperfusion injury (MIRI) caused by the reperfusion therapy of myocardial ischemic diseases is a kind of major disease that threatens human health and lives severely. There are lacking of effective therapeutic measures for MIRI. MicroRNAs (miRNAs) are abundant in mammalian species and play a critical role in the initiation, promotion, and progression of MIRI. However, the biological role and molecular mechanism of miRNAs in MIRI are not entirely clear.

Methods: We used bioinformatics analysis to uncover the significantly different miRNA by analyzing transcriptome sequencing data from myocardial tissue in the mouse MIRI model. Multiple miRNA-related databases, including miRdb, PicTar, and TargetScan were used to forecast the downstream target genes of the differentially expressed miRNA. Then, the experimental models, including male C57BL/6J mice and HL-1 cell line, were used for subsequent experiments including quantitative real-time polymerase chain reaction analysis, western blot analysis, hematoxylin and eosin staining, flow cytometry, luciferase assay, gene interference, and overexpression.

Results: MiR-582-5p was found to be differentially upregulated from the transcriptome sequencing data. The elevated levels of miR-582-5p were verified in MIRI mice and hypoxia/reperfusion (H/R)-induced HL-1 cells. Functional experiments revealed that miR-582-5p promoted apoptosis of H/R-induced HL-1 cells via downregulating cAMP-response element-binding protein 1 (Creb1). The inhibiting action of miR-582-5p inhibitor on H/R-induced apoptosis was partially reversed after Creb1 interference.

Conclusions: Collectively, the research findings reported that upregulation of miR-582-5p promoted H/R-induced cardiomyocyte apoptosis by inhibiting Creb1. The potential diagnostic and therapeutic strategies targeting miR-582-5p and Creb1 could be beneficial for the MIRI treatment.

Keywords: Creb1; apoptosis; miR-582-5p; myocardial ischemia-reperfusion injury.

Figure 1

Expression of miR‐582‐5p and its target gene, Creb1, in mice MIRI models. (A) The heatmap of the gene expression of the seven significantly differentially expressed miRNAs. (B) ECG presentation in the sham and MIRI groups. (C) HE staining of myocardial tissue in the sham and MIRI groups. (D) Expression of the seven differentially expressed miRNAs in the mouse MIRI model. (E) Target gene prediction for miR‐582‐5p. (F) The binding sites between miR‐582‐5p and Creb1 were predicted on the starBase website. (G) The mRNA expression of Creb1 in the MIRI model. (H) The protein expression of Creb1 in the MIRI model. Data were expressed as the mean ± SD. 3′‐UTR, 3′‐untranslated region; Creb1, cAMP‐response element binding protein 1; ECG, electrocardiograph; HE, hematoxylin and eosin; H/R, hypoxia/reperfusion; MIRI, myocardial ischemia–reperfusion injury; miRNA, microRNA.

Figure 2

MiR‐582‐5p directly targets Creb1 in H/R‐induced cardiomyocytes. (A) Expression of the seven miRNAs in H/R‐induced cardiomyocytes. (B) Expression of Creb1 in H/R‐induced cardiomyocyte. (C) Expression of miR‐582‐5p after transfection of mimic and inhibitor of miR‐582‐5p, respectively. (D) Expression of Creb1 mRNA after transfection of mimic and inhibitor of miR‐582‐5p, respectively. (E, F) Expression of Creb1 protein after transfection of mimic and inhibitor of miR‐582‐5p, respectively. (G) Schematic representation of the pmirGLO plasmid vector. (H) Relative luciferase activity of Creb1‐Wt/Mut was examined by luciferase reporter assay in HL‐1 cells. The images are representative of three independent experiments per group and data. Creb1, cAMP‐response element binding protein 1; H/R, hypoxia/reperfusion; miRNA, microRNA; mRNA, messenger RNA; Mut, mutant; NC, negative control; Wt, wild‐type.

Figure 3

Overexpressed Creb1 inhibited H/R‐induced cardiomyocyte apoptosis. (A, B) The apoptosis rate was determined by using flow cytometry at 48 h. (C–G) Protein extracts from cells were immunoblotted with apoptosis‐related protein antibodies. β‐actin was a loading control. The quantification of Creb1 (D), Bcl‐2 (E), cleaved‐caspase 3 (F), and Bax (G) were quantified by use of FIJI software. Data are present as mean ± SD. Creb1, cAMP‐response element binding protein 1; H/R, hypoxia/reperfusion; NC, negative control.

Figure 4

The inhibitory action of miR‐582‐5p knockdown in H/R‐induced cardiomyocytes was abolished by the downregulation of Creb1. (A, B) The apoptosis rate was determined by using flow cytometry at 48 h. (C–G) Protein extracts from cells were immunoblotted with apoptosis‐related protein antibodies. β‐actin was a loading control. The quantification of Creb1 (D), Bcl‐2 (E), cleaved‐caspase 3 (F), and Bax (G) were quantified by use of FIJI software. Data are present as mean ± SD. Creb1, cAMP‐response element binding protein 1; H/R, hypoxia/reperfusion; NC, negative control; siRNA, small interfering RNA.