Korean Circ J.  2024 May;54(5):233-252. 10.4070/kcj.2023.0131.

LncRNA PART1 Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating TFAP2C/DUSP5 Axis via miR-302a-3p

• Zeng M
• Wei X
• Zhou J
• Luo S

• KMID: 2556520
• DOI: http://doi.org/10.4070/kcj.2023.0131

Abstract

Background and Objectives
Myocardial ischemia-reperfusion injury (MIRI) refers to the damage of cardiac function caused by restoration of blood flow perfusion in ischemic myocardium. However, long non-coding RNA prostate androgen regulated transcript 1 (PART1)’s role in MIRI remain unclear.
Methods
Immunofluorescence detected LC3 expression. Intermolecular relationships were verified by dual luciferase reporter assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and transferase-mediated dUTP nick-end labeling (TUNEL) assays analyzed cell viability and apoptosis. The release of lactate dehydrogenase was tested via enzyme-linked immunosorbent assay (ELISA). Left anterior descending coronary artery surgery induced a MIRI mouse model. Infarct area was detected by 2,3,5-triphenyltetrazolium chloride staining. Hematoxylin and eosin staining examined myocardial injury. ELISA evaluated myocardial marker (creatine kinase MB) level.
Results
PART1 was decreased in hypoxia/reoxygenation (H/R) induced AC16 cells and MIRI mice. PART1 upregulation attenuated the increased levels of Bax, beclin-1 and the ratio of LC3II/I, and enhanced the decrease of Bcl-2 and p62 expression in H/R-treated cells. PART1 upregulation alleviated H/R-triggered autophagy and apoptosis via miR-302a-3p. Mechanically, PART1 targeted miR-302a-3p to upregulate transcription factor activating enhancer-binding protein 2C (TFAP2C). TFAP2C silencing reversed the protected effects of miR-302a-3p inhibitor on H/R treated AC16 cells. We further established TFAP2C combined to dual-specificity phosphatase 5 (DUSP5) promoter and activated DUSP5. TFAP2C upregulation suppressed H/R-stimulated autophagy and apoptosis through upregulating DUSP5. Overexpressed PART1 reduced myocardial infarction area and attenuated MIRI in mice.
Conclusion
PART1 improved the autophagy and apoptosis in H/R-exposed AC16 cells through miR-302a-3p/TFAP2C/DUSP5 axis, which might provide novel targets for MIRI treatment.

Keyword

LncRNA; Ischemia; Reperfusion

Figure

• 

  Figure 1 Overexpression of PART1 inhibited autophagy and apoptosis in H/R-challenged cardiomyocytes. (A) MTT assay detected cell viability at different hypoxia time points. (B) PART1 transfection efficiency was verified by RT-qPCR. (C) RT-qPCR detection of PART1 level after overexpression of PART1 in H/R-treated AC16 cells. (D) MTT tested cell viability. (E) ELISA tested LDH concentration. (F) Cells apoptosis was detected utilizing Flow cytometry. (G) TUNEL staining detected apoptosis. (H) Bcl-2 and Bax expressions were measured using Western blot. (I) Immunofluorescence detection of LC3 expression in AC16 cells. (J) Western blot detected p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; oe- = overexpression plasmids; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TUNEL = transferase-mediated dUTP nick-end labeling.*p<0.05, **p<0.01, ***p<0.001.

• 

  Figure 2 PART1 targeted miR-302a-3p and upregulated TFAP2C level. (A) StarBase predicted binding site of miR-302a-3p with PART1 or TFAP2C. (B) Luciferase activity of PART1-WT/MUT (left panel) or TFAP2C-WT/MUT (right panel) in AC16 cells treated with miR-302a-3p mimics or mimics NC. (C, D) RT-qPCR assessed miR-302a-3p (C) and TFAP2C (D) levels after overexpression of PART1 in AC16 cells. (E) Western blot analyzed TFAP2C protein level. (F, G) RT-qPCR detected miR-302a-3p (F) and TFAP2C (G) after miR-302a-3p overexpression or inhibition in AC16 cells. (H) Western blot tested the protein level of TFAP2C. Data were shown as mean ± SD based on three independent experiments (n=3).MUT = mutant type; NC = negative control; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; WT = wild type.*p<0.05, **p<0.01, ***p<0.001.

• 

  Figure 3 PART1 participated in H/R-mediated cardiomyocyte function injury via regulating miR-302a-3p. (A) RT-qPCR determined miR-302a-3p level. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry analyzed cell apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot tested Bcl-2 and Bax levels. (G) Immunofluorescence measured LC3 expression in AC16 cells. (H) Western blot detected the protein levels of p62, LC3II/I, Beclin-1. Data were shown as mean ± SD based on three independent experiments (n=3).ELISA = enzyme-linked immunosorbent assay; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TUNEL = transferase-mediated dUTP nick-end labeling.*p<0.05, **p<0.01, ***p<0.001.

• 

  Figure 4 MiR-302a-3p inhibitor attenuated H/R-triggered cardiomyocyte autophagy and apoptosis through TFAP2C. (A) TFAP2C transfection efficiency was verified by RT-qPCR and Western blot. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry determined apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot analyzed Bcl-2 and Bax expression levels. (G) Immunofluorescence detected LC3 expression in AC16 cells. (H) Western blot measured p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; TUNEL = transferase-mediated dUTP nick-end labeling.*p<0.05, **p<0.01, ***p<0.001.

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  Figure 5 TFAP2C bound to DUSP5 promoter and upregulated DUSP5. (A) Binding site between TFAP2C and DUSP5 by JASPAR. (B) Dual luciferase reporter assay verified the binding of TFAP2C to DUSP5 promoter. (C, D) RT-qPCR and Western blot detected the expression of TFAP2C and DUSP5 after overexpression of TFAP2C. Data were shown as mean ± SD based on three independent experiments (n=3).DUSP5 = dual-specificity phosphatase 5; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C.*p<0.05, **p<0.01, ***p<0.001.

• 

  Figure 6 TFAP2C upregulation suppressed H/R-challenged cardiomyocyte autophagy and apoptosis through regulating DUSP5. (A) RT-qPCR and Western blot analysis of DUSP5 expression. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry detected apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot analyzed Bcl-2 and Bax expression levels. (G) Immunofluorescence detected LC3 in AC16 cells. (H) Western blot detected p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).DUSP5 = dual-specificity phosphatase 5; ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; TUNEL = transferase-mediated dUTP nick-end labeling.*p<0.05, **p<0.01, ***p<0.001.

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  Figure 7 Enforced expression of PART1 relieved I/R-induced MIRI. A mice model of MIRI was prepared and treated with a PART1 overexpression vector. (A) Mouse MIRI model construction timeline. (B) TTC staining detected myocardial infarct volume. (C) H&E staining detected myocardial histopathological changes in mice. (D) Blood level of myocardial marker CK-MB was measured by ELISA. (E) RT-qPCR detection of PART1 expression in mouse myocardial tissue. (F) TUNEL staining detected cardiomyocyte apoptosis. (G) Cleaved caspase3 expression in mouse myocardial tissue was assessed with IHC. (H) Western blot detected p62, LC3II/I, Beclin-1 levels. Data are the means ± SD for three independent experiments (n=5 mice/group).CK-MB = creatine kinase MB; ELISA = enzyme-linked immunosorbent assay; H&E = hematoxylin and eosin; IHC = immunohistochemistry; I/R = ischemia/reperfusion; MIRI = myocardial ischemia-reperfusion injury; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TTC = 2,3,5-Triphenyltetrazolium chloride; TUNEL = transferase-mediated dUTP nick-end labeling.*p<0.05, **p<0.01, ***p<0.001.

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