MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury

doi:10.12122/j.issn.1673-4254.2024.06.19

Abstract

Abstract:

Objectives To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injury. Methods Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC) to measure miRNA-224-5p levels and other biochemical parameters. In cultured H9c2 cells with H/R injury, the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability, malondialdehyde (MDA) content, and superoxide dismutase 2 (SOD2) and lactate dehydrogenase (LDH) activities were tested. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN. Bioinformatics methods were used to analyze the potential mechanisms of the target genes. The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR, the protein expressions of PTEN, Bcl-2, Bax, cleaved caspase-3, SOD2, p-PI3K/PI3K, p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting, and cell apoptosis was analysed with flow cytometry. Results The levels of blood glucose, C-reactive protein, CK, CK-MB and cTnI were significantly higher in the AMI group compared with the HC group (P<0.05). The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury. The viability of H9c2 cells decreased time-dependently following H/R injury. PTEN was a target gene of miR-224-5p, and the PI3K/Akt pathway was the most significantly enriched pathway. H9c2 cells with H/R injury showed significantly decreased SOD2 activity, increased LDH activity and MDA content, increased cell apoptosis, decreased protein expression levels of p-PI3K, p-Akt, p-FoxO1, SOD2, and Bcl-2, and increased expressions of PTEN, Bax, and cleaved caspase-3. These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure. Conclusion MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells.

Key words: hypoxia/reoxygenation, microRNAs, oxidative stress, apoptosis

Guoxin LIANG, Hongyue TANG, Chang GUO, Mingming ZHANG. MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury[J]. Journal of Southern Medical University, 2024, 44(6): 1173-1181.

Figures/Tables 6

Tab.1 Clinical parameters of the participants

Clinical parameters	STEMI (n=80)	NSTEMI (n=80)	HC (n=80)	P₁	P₂	P₃
Age (year)	60.5±11.46	58.58±12.19	59.24±12.85	0.514	0.739	0.306
Gender (male/female)	44/36	41/39	43/37	0.950	0.874	0.635
Hypertension	75% (60)	72% (58)	0	≠	≠	0.720
Tobacco	70% (56)	65% (52)	50% (40)	0.001	0.055	0.971
Diabetes mellitus	40% (32)	30% (24)	0	≠	≠	0.185
Hypercholesterolemia	80% (64)	75% (60)	0	≠	≠	0.449
Body mass index (kg/m²)	25.2±4.2	24.5±3.4	22.4±1.8	0.795	0.839	0.973
Previous MI	10% (8)	6% (4)	0	≠	≠	0.378
Previous PTCA	6	4	0	≠	≠	0.744
Previous CABG	5% (4)	2% (2)	0	≠	≠	0.701
C-reactive protein (mg/L)	18.17±5.52	18.51±4.86	2.13±1.36	<0.05	<0.005	0.680
Glucose (mmol/L)	5.75±1.85	5.68±1.82	4.81±0.48	<0.05	<0.05	0.810
CK(U/L)	483.9±57.27	474.9±50.49	127.29±26.33	<0.05	<0.05	0.293
CK-MB(U/L)	76.43±23.85	73.71±20.68	9.82±4.88	<0.05	<0.05	0.472
cTnI (mg/mL)	163.66±21.50	160.73±23.88	26.40±13.77	<0.05	<0.05	0.403

Fig.1 Expression level of miR-224-5p and its clinical diagnostic value. A: High-throughput data analysis. B: miR-224-5p expression levels.*P<0.05 vs HC group; C: AUC of HC vs STEMI; D: AUC of HC vs NSTEMI.

Fig.2 Hypoxia/reoxygenation decreases viability and promotes apoptosis of cultured cardiomyocytes. A, B: MTT colorimetric assay to determine cell viability (Mean±SD, n=6). C: Western blotting for detecting protein expressions of cleaved caspase3, Bcl-2, and Bax. D-F: Relative expressions of Bcl2/Bax proteins, cleaved caspase3 and miR-224-5p in H9c2 cells in each group (Mean±SD, n=3). compared with Control group, *P<0.05; ##P<0.05 vs Hypoxia (12 h) group.

Fig3 Target gene of miR-224-5p and KEGG enrichment analysis. A: MiR-224-5p gene regulatory network. B: Binding sequences of PTEN mRNA 3'-UTR and miR-224-5p. C: PTEN-Wt or PTEN- Mut reporter Vector cotransfected into H9C2 cells with vehicle (control), miR-224-5p mimic, or negative control (miRNA-NC). Luciferase activity was normalized to the activity in the control group. Data are presented as Mean±SD (n=3). D: KEGG pathway enrichment analysis. *P<0.05 vs miR-NC group.

Fig.4 Levels of LDH, MDA, and SOD2 in H9c2 cells in each group (Mean±SD, n=6). A: LDH activity. B: MDA content. C: SOD2 activity measurement. *P<0.05 vs Control group; ##P<0.05 vs H/R+miR-NC group.

Fig5 Effect of overexpression of miR-224-5p on hypoxia/hypoxia-induced oxidative stress and apoptotic proteins in H9c2 cells in each group (Mean±SD, n=3). A, B: Western blotting for detecting oxidative stress pathway protein expressions. C, D: Western blotting for detecting cleaved caspase-3, Bax and Bcl-2 protein expressions. E, F: Flow cytometry for analyzing apoptosis rate in H9c2 cells in each group (Mean±SD, n=3); *P<0.05 compared with Control group; #P<0.05 vs H/R+ miR-NC group, ##P<0.05.

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