Korean Circ J.  2023 Mar;53(3):151-167. 10.4070/kcj.2022.0242.

LncRNA AC005332.7 Inhibited Ferroptosis to Alleviate Acute Myocardial Infarction Through Regulating miR-331-3p/CCND2 Axis

• Dai R
• Yang X
• He W
• Su Q
• Deng X
• Li J

• KMID: 2540393
• DOI: http://doi.org/10.4070/kcj.2022.0242

Abstract

Background and Objectives
Acute myocardial infarction (AMI) often occurs suddenly and leads to fatal consequences. Ferroptosis is closely related to the progression of AMI. However, the specific mechanism of ferroptosis in AMI remains unclear.
Methods
We constructed a cell model of AMI using AC16 cells under oxygen and glucose deprivation (OGD) conditions and a mice model of AMI using the left anterior descending (LAD) ligation. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide was employed to determine cell viability. The levels of lactate dehydrogenase, creatine kinase, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and iron were measured using corresponding kits. Dual luciferase reporter gene assay, RNAbinding protein immunoprecipitation, and RNA pull-down were performed to validate the correlations among AC005332.7, miR-331-3p, and cyclin D2 (CCND2). Hematoxylin and eosin staining was employed to evaluate myocardial damage.
Results
AC005332.7 and CCND2 were lowly expressed, while miR-331-3p was highly expressed in vivo and in vitro models of AMI. AC005332.7 sufficiency reduced ROS, MDA, iron, and ACSL4 while boosting the GSH and GPX4, indicating that AC005332.7 sufficiency impeded ferroptosis to improve cardiomyocyte injury in AMI. Mechanistically, AC005332.7 interacted with miR-331-3p, and miR-331-3p targeted CCND2. Additionally, miR-331-3p overexpression or CCND2 depletion abolished the suppressive impact of AC005332.7 on ferroptosis in OGD-induced AC16 cells. Moreover, AC005332.7 overexpression suppressed ferroptosis in mice models of AMI.
Conclusions
AC005332.7 suppressed ferroptosis in OGD-induced AC16 cells and LAD ligation-operated mice through modulating miR-331-3p/CCND2 axis, thereby mitigating the cardiomyocyte injury in AMI, which proposed novel targets for AMI treatment.

Keyword

Myocardial infarction; lncRNA; MicroRNA; Cyclin D2; Ferroptosis

Figure

• 

  Figure 1 OGD reduced AC005332.7 expression and promoted ferroptosis in AC16 cells. (A) qRT-PCR was applied to detect AC005332.7 expression. (B) MTT was used for evaluating cell viability. (C-G) LDH, ROS, GSH, MDA, and iron levels were detected using corresponding kits and methods. (H) Western blot was used for examining GPX4 and ACSL4 expression. Data were obtained from 3 independent experiments. Data were shown as mean±standard deviation (n=3).ACSL4 = acyl-CoA synthetase long chain family member 4; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GSH = glutathione; LDH = lactate dehydrogenase; GPX4 = glutathione peroxidase 4; MDA = malondialdehyde; MTT = 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 2 AC005332.7 repressed ferroptosis in OGD-induced AC16. (A) qRT-PCR was applied to detect AC005332.7 expression. (B) qRT-PCR was used for detecting AC005332.7 expression. (C) MTT was employed for evaluating cell viability. (D-H) LDH, ROS, GSH, MDA, and iron levels were examined using corresponding kits and methods. (I) Western blot was conducted to investigate GPX4 and ACSL4 expression. Data were shown as mean±standard deviation (n=3).ACSL4 = acyl-CoA synthetase long-chain family member 4; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GPX4 = glutathione peroxidase 4; GSH = glutathione; LDH = lactate dehydrogenase; MDA = malondialdehyde; MTT = 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide; NC = negative control; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 3 AC005332.7 indirectly mediated CCND2 expression through sponging miR-331-3p. (A, B) qRT-PCR was applied for detecting miR-331-3p and CCND2 expression. (C) Western blot was conducted to examine CCND2 expression. (D) qRT-PCR was used to determine miR-331-3p expression in AC16 cells transfected with miR-331-3p mimics and inhibitors. (E) Starbase predicted the potential binding site between AC005332.7 and miR-331-3p. (F) A dual luciferase reporter gene assay was conducted to evaluate luciferase activity. (G) RIP was used to validate the interaction between AC005332.7 and miR-331-3p. (H) qRT-PCR was employed to detect miR-331-3p expression. (I) qRT-PCR was used to examine miR-331-3p expression. (J) Starbase predicted the potential binding site between miR-331-3p and CCND2. (K) A dual luciferase reporter gene assay was conducted to evaluate luciferase activity. (L) RNA-pull down was employed for validating the interaction between miR-331-3p and CCND2. (M-R) qRT-PCR and western blot were applied to measure CCND2 expression. Data were shown as mean±standard deviation (n=3).CCND2 = cyclin D2; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IgG = immunoglobulin G; MUT = mutant; NC = negative control; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; RIP = RNA-binding protein immunoprecipitation; WT = wild type.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 4 AC005332.7 inhibited ferroptosis in OGD-induced AC16 cells via reducing miR-331-3p expression. (A) qRT-PCR was applied for detecting miR-331-3p expression. (B) MTT was conducted to evaluate cell viability. (C-G) The LDH, ROS, GSH, MDA, and iron levels were tested using corresponding kits and methods. (H) Western blot was applied to examine GPX4 and ACSL4 expression. Data were shown as mean±standard deviation (n=3).ACSL4 = acyl-CoA synthetase long-chain family member 4; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GPX4 = glutathione peroxidase 4; GSH = glutathione; LDH = lactate dehydrogenase; MDA = malondialdehyde; MMT = 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide; NC = negative control; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 5 AC005332.7 impeded ferroptosis in OGD-induced AC16 via elevating CCND2 expression. (A, B) qRT-PCR and western blot were conducted to detect CCND2 expression. (C, D) qRT-PCR and western blot were used for detecting CCND2 expression. (E) MTT was conducted to evaluate cell viability. (F-J) The LDH, ROS, GSH, MDA, and iron levels were investigated using corresponding kits and methods. (K) Western blot was applied to examine GPX4 and ACSL4 expression. Data were shown as mean±standard deviation (n=3).ACSL4 = acyl-CoA synthetase long chain family member 4; CCND2 = cyclin D2; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GPX4 = glutathione peroxidase 4; GSH = glutathione; LDH = lactate dehydrogenase; MDA = malondialdehyde; MTT = 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide; NC = negative control; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 6 AC005332.7 overexpression restrained ferroptosis in an animal model of AMI. (A) qRT-PCR was performed to detect AC005332.7 expression. (B) Western blot was used for measuring CCND2 expression. (C) H&E staining was conducted to evaluate myocardial damage. (D) Immunohistochemical staining examined the expression of Ki67, CCND2, and GPX4. (E-J) LDH activity and CK, ROS, GSH, MDA, and iron levels were evaluated using corresponding kits and methods. (K) Western blot was applied to examine GPX4 and ACSL4 expression. Data were shown as mean ± standard deviation (n=9).ACSL4 = acyl-CoA synthetase long-chain family member 4; AMI = acute myocardial infarction; CCND2 = cyclin D2; CK = creatine kinase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GPX4 = glutathione peroxidase 4; GSH = glutathione; H&E = hematoxylin and eosin; LDH = lactate dehydrogenase; MDA = malondialdehyde; MTT = 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide; NC = negative control; OGD = oxygen and glucose deprivation; qRT-PCR = quantitative reverse transcription polymerase chain reaction; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

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