miR-224-5p调控PI3K/Akt/FoxO1轴抑制氧化应激减轻缺氧/复氧诱导的心肌细胞损伤

doi:10.12122/j.issn.1673-4254.2024.06.19

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1173-1181.doi: 10.12122/j.issn.1673-4254.2024.06.19

• • 上一篇

miR-224-5p调控PI3K/Akt/FoxO1轴抑制氧化应激减轻缺氧/复氧诱导的心肌细胞损伤

梁国新¹^,²^,³(), 唐红悦³^,⁴, 郭畅²^,³, 张明明³

^1.南京医科大学附属南京明基医院检验科，江苏南京 210000
^2.华北理工大学研究生学院，河北唐山 063000
^3.河北省人民医院临床医学研究中心河北省代谢病重点实验室，河北石家庄 050051
^4.河北北方学院研究生学院，河北张家口 075132

收稿日期:2024-01-03 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 张明明 E-mail:lgx202309@163.com
作者简介:梁国新，硕士，住院医师，E-mail: lgx202309@163.com
基金资助:
河北省政府资助临床医学人才培养项目(2020009);河北省自然科学基金资助项目(H2024307005)

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

Guoxin LIANG¹^,²^,³(), Hongyue TANG³^,⁴, Chang GUO²^,³, Mingming ZHANG³

^1.Department of Laboratory Medicine, Nanjing BenQ Medical Center, Affiliated BenQ Hospital of Nanjing Medical University, Nanjing 210000, China
^2.Graduate School, North China University of Science and Technology, Tangshan 063000, China
^3.Hebei Provincial Key Laboratory of Metabolic Diseases, Medical Research Center, Hebei General Hospital, Shijiazhuang 050051, China
^4.Graduate School of Hebei North College, Zhangjiakou 075132, China

Received:2024-01-03 Online:2024-06-20 Published:2024-07-01
Contact: Mingming ZHANG E-mail:lgx202309@163.com

摘要/Abstract

摘要：

目的探究miRNA-224-5p在缺氧/复氧（H/R）诱导心肌细胞损伤中的作用机制。方法收集160 例急性心肌梗死（AMI）患者和80 例健康体检者（HC）的血浆检测miRNA-224-5p及生化指标。H9c2细胞分为对照组（Control）、H/R组、H/R+miR-224-5pNC组、H/R+miR-224-5p mimics组。噻唑蓝（MTT）检测细胞活力；试剂盒检测丙二醛（MDA）、超氧化物歧化酶2（SOD2）和乳酸脱氢酶（LDH）；双荧光素酶报告基因实验验证miR-224-5p与PTEN的靶向关系；生物信息学方法对靶基因的潜在机制进行分析；qRT-PCR检测miRNA-224-5p mRNA表达；Western blotting检测PTEN、Bcl-2、Bax、Cleaved caspase-3、SOD2、p-PI3K/PI3K、p-Akt/Ak及p-FoxO1/FoxO1蛋白水平；流式细胞术检测细胞凋亡率。结果与 HC 组相比，AMI组的血糖、 C 反应蛋白、CK、CK-MB 和cTnI水平均显著高于对照组（P<0.05）。AMI组和H/R组miR-224-5p的表达低于对照组（P<0.05）；心肌细胞活力呈缺氧/复氧时间依赖性减少；PTEN是miR-224-5p的靶基因；PI3K/Akt通路是最显著富集的通路；与Control组相比，H/R组SOD2的活性降低，LDH的活性和MDA的含量均上升，细胞凋亡率上升（P<0.05），细胞中p-PI3K、p-Akt、p-FoxO1、SOD2和Bcl-2蛋白表达水平均降低，PTEN、Bax和Cleaved caspase-3蛋白表达均升高（P<0.05）；与H/R组比较，H/R+miR-224-5p mimics组SOD2的活性显著上升，LDH、MDA的水平和细胞凋亡率均显著降低（P<0.05），细胞中p-PI3K、p-Akt、p-FoxO1、SOD2和Bcl-2的表达均上调，PTEN、Bax和Cleaved caspase3蛋白表达水平均降低（P<0.05）。结论 miR-224-5p过表达通过PI3K/Akt/FoxO1轴上调抗氧化基因SOD2的表达，缓解H/R诱导的H9c2细胞的氧化应激，减少细胞凋亡。

关键词: 缺氧/复氧, 微小RNA, 氧化应激, 凋亡

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

梁国新, 唐红悦, 郭畅, 张明明. miR-224-5p调控PI3K/Akt/FoxO1轴抑制氧化应激减轻缺氧/复氧诱导的心肌细胞损伤[J]. 南方医科大学学报, 2024, 44(6): 1173-1181.

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.

图/表 6

表1 研究对象的临床参数

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

图1 miR-224-5p的表达水平及临床诊断价值

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.

图2 缺氧/复氧降低心肌细胞活力并促进凋亡

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.

图3 miR-224-5p靶基因和KEGG富集分析

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.

图4 各实验组LDH、MDA和SOD2的水平

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.

图 5 miR-224-5p的过表达对缺氧/缺氧诱导的氧化应激和细胞凋亡蛋白的影响

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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miR-224-5p调控PI3K/Akt/FoxO1轴抑制氧化应激减轻缺氧/复氧诱导的心肌细胞损伤

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

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