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.
CONCLUSIONS: 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.
方法:从160例急性心肌梗死患者和80例健康对照(HC)中收集血浆样本,以测量miRNA-224-5p水平和其他生化参数。在培养的H/R损伤的H9c2细胞中,用miR-224-5p模拟物或阴性对照序列转染对细胞活力的影响,丙二醛(MDA)含量,并检测了超氧化物歧化酶2(SOD2)和乳酸脱氢酶(LDH)的活性。进行双荧光素酶报告基因测定以验证miR-224-5p与PTEN之间的靶向关系。生物信息学方法用于分析靶基因的潜在机制。qRT-PCR检测miRNA-224-5p在处理细胞中的表达,PTEN的蛋白质表达,Bcl-2,Bax,caspase-3,SOD2,p-PI3K/PI3K,使用蛋白质印迹法测定p-Akt/Ak和p-FoxO1/FoxO1,用流式细胞仪分析细胞凋亡。
结果:血糖水平,C反应蛋白,CK,AMI组CK-MB和cTnI明显高于HC组(P<0.05)。miR-224-5p的表达水平在STEMI和NSTEMI患者以及H/R损伤的H9c2细胞中显著降低。H/R损伤后,H9c2细胞的活力随时间而降低。PTEN是miR-224-5p的靶基因,而PI3K/Akt途径是最显著的富集途径。H/R损伤的H9c2细胞显示SOD2活性显著降低,LDH活性和MDA含量增加,细胞凋亡增加,p-PI3K蛋白表达水平降低,p-Akt,p-FoxO1,SOD2和Bcl-2,以及PTEN的表达增加,Bax,和裂解的caspase-3。通过在H/R暴露之前用miR-224-5p模拟物转染细胞,这些变化明显减弱。
结论:MiR-224-5p过表达可通过PI3K/Akt/FoxO1轴上调抗氧化基因SOD2的表达,从而减轻H/R诱导的H9c2细胞氧化应激,减少细胞凋亡。