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Long non-coding RNA-AK138945 regulates myocardial ischemia-reperfusion injury via the miR-1-GRP94 signaling pathway
doi: 10.2478/fzm-2024-0004
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Abstract:
Objective Myocardial ischemia-reperfusion injury (MIRI) is one of the leading causes of death from cardiovascular disease in humans, especially in individuals exposed to cold environments. Long non-coding RNAs (lncRNAs) regulate MIRI through multiple mechanisms.This study explored the regulatory effect of lncRNA-AK138945 on myocardial ischemia-reperfusion injury and its mechanism. Methods In vivo, 8- to 12-weeks-old C57BL/6 male mice underwent ligation of the left anterior descending coronary artery for 50 minutes followed by reperfusion for 48 hours. In vitro, the primary cultured neonatal mouse ventricular cardiomyocytes (NMVCs) were treated with 100 μmol/L hydrogen peroxide (H2O2). The knockdown of lncRNA-AK138945 was evaluated to detect cardiomyocyte apoptosis, and a glucose-regulated, endoplasmic reticulum stress-related protein 94 (GRP94) inhibitor was used to detect myocardial injury. Results We found that the expression level of lncRNA-AK138945 was reduced in MIRI mouse heart tissue and H2O2-treated cardiomyocytes. Moreover, the proportion of apoptosis in cardiomyocytes increased after lncRNA-AK138945 was silenced. The expression level of Bcl2 protein was decreased, and the expression level of Bad, Caspase 9 and Caspase 3 protein was increased. Our further study found that miR-1a-3p is a direct target of lncRNA-AK138945, after lncRNA-AK138945 was silenced in cardiomyocytes, the expression level of miR-1a-3p was increased while the expression level of its downstream protein GRP94 was decreased. Interestingly, treatment with a GRP94 inhibitor (PU-WS13) intensified H2O2-induced cardiomyocyte apoptosis. After overexpression of FOXO3, the expression levels of lncRNA-AK138945 and GRP94 were increased, while the expression levels of miR-1a-3p were decreased. Conclusion LncRNA-AK138945 inhibits GRP94 expression by regulating miR-1a-3p, leading to cardiomyocyte apoptosis. The transcription factor Forkhead Box Protein O3 (FOXO3) participates in cardiomyocyte apoptosis induced by endoplasmic reticulum stress through up-regulation of lncRNA-AK138945. -
Key words:
- myocardial ischemia reperfusion /
- lncRNA /
- apoptosis /
- microRNA GRP94
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Figure 1. Expression of lncRNA-AK138945 in a mouse model of myocardial ischemia-reperfusion injury (MIRI). (A) Significant decreases in ejection fraction (EF) and fractional shortening (FS) in MIRI mice compared to the control group, indicating the successful establishment of the MIRI model. ** vs. Sham, P < 0.01, Sham, N = 6; MIRI, N = 4. (B-D) Western blot analysis on the expression levels of apoptotic proteins Caspase 9, Caspase 3, and Bad in myocardial tissue. * vs. Sham, P < 0.05; ** vs. Sham, P < 0.01, N = 3-5. (E) qPCR analysis on the expression of lncRNA-AK138945 in neonatal mouse cardiomyocytes after hydrogen peroxide treatment * vs. Control, P < 0.05; ** vs. Control, P < 0.01, N = 3. (F) Changes of lncRNA-AK138945 expression in cardiac tissue of MIRI model. * vs. Sham, P < 0.05; ** vs. Sham, P < 0.01; *** vs. Sham, P < 0.001, N = 3-4.
Figure 2. LncRNA-AK138945 knockdown facilitates cardiomyocyte apoptosis. (A) Verification of lncRNA-AK138945 knockdown efficiency by its siRNA (siAK138945)** vs. Control, P < 0.01; ### vs. +siAK138945, P < 0.001, N = 3. (B) Reduced cardiomyocyte viability by siAK138945, as measured by CCK8 assay. *** vs. Control, P < 0.001; ### vs. +siAK138945, P < 0.001, N = 6. (C) siAK138945 increased cardiomyocyte apoptosis by siAK138945 as unveiled by Tunel staining. ** vs. Control, P < 0.01; ## vs. +siAK138945, P < 0.01, N = 3. (D) siAK138945 increased cardiomyocyte death, as demonstrated by survival/death (Live/Dead) staining. Red fluorescence represents dead cells, and green fluorescence represents live cells. * vs. Control, P < 0.05; # vs. +siAK138945, P < 0.05, N = 3.
Figure 3. LncRNA-AK138945 knockdown anomaly alters the expression of apoptosis-related proteins in cardiomyocytes. (A-C) LncRNA-AK138945 knockdown by siAK138945 increases the expression levels of apoptotic proteins Caspase3, Caspase9, and Bad in cardiomyocytes. * vs. Control, P < 0.05; ** vs. Control, P < 0.01; *** vs. Control, P < 0.001; # vs. +siAK138945, P < 0.05; ## vs. +siAK138945, P < 0.01, N = 3-4. (D) siAK138945 significantly reduced the expression of Bcl2. *** vs. Control, P < 0.001; # vs. +siAK138945, P < 0.05, N = 5.
Figure 4. Experimental verification of miR-1a-3p as a downstream target of lncRNA-AK138945. (A) The predicted binding sites between miR-1a-3p and lncRNA-AK138945. (B) Luciferase reporter gene activity. *** vs. NC, P < 0.001, N = 3. (C) Upregulation of miR-1a-3p expression following lncRNA-AK138945 knockdown. * vs. +siNC, P < 0.05; ** vs. +siNC, P < 0.01, N = 3-6. (D-E) Downregulation of GRP94 expression post-lncRNA-AK138945 knockdown. ** vs. Control, P < 0.01; *** vs. Control, P < 0.001; # vs. +siAK138945, P < 0.05; ## vs. +siAK138945, P < 0.01, N = 5-6.
Figure 5. Inhibition of GRP94 induces cardiomyocyte apoptosis. (A-C) After treatment with a GRP94 inhibitor, the expression of apoptosis-related proteins Caspase3, Caspase9 and Bad was increased. *** vs. Control, P < 0.001; # vs. H2O2, P < 0.05, ## vs. H2O2, P < 0.01, N = 3-6. (D) The expression of anti-apoptotic protein Bcl2 was reduced after treatment with GRP94 inhibitor. ** vs. Control, P < 0.01; # vs. H2O2, P < 0.05, N = 6.
Figure 6. Effect of transcription factor FOXO3 on the AK138945-miR-1-GRP94 signaling pathway. (A) The expression of FOXO3 in myocardial tissue. *** vs. Sham, P < 0.001, Sham, N = 4; MIRI, N = 4. (B) Verification of FOXO3 overexpression efficiency.*** vs. Control, P < 0.001; # vs. FOXO3, P < 0.05, N = 4. (C-D) The expression levels of lncRNA-AK138945 and GRP94 are up-regulated after FOXO3 overexpression. * vs. Control, P < 0.05; *** vs. Control, P < 0.001; # vs. FOXO3, P < 0.05; ## vs. FOXO3, P < 0.01, N = 6. (E) FOXO3 overexpression downregulates the expression level of miR-1a-3p. ** vs. Control, P < 0.01; # vs. FOXO3, P < 0.05, N = 6.
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