Background
This article aims to use high-throughput sequencing to identify miRNAs associated with ferroptosis in myocardial ischemia-reperfusion injury, select a target miRNA, and investigate its role in H9C2 cells hypoxia-reoxygenation injury.
Conclusion
In summary, miRNA-541-5p may be a biomarker of myocardial I/R damage diseases and can regulate oxidative stress and iron death by inhibiting the expression of miRNA-541-5p, thereby reducing mechanisms of I/R injury.
Methods
SD rats and H9C2 cells were used as subjects. ELISA kits quantified MDA, SOD, GSH, LDH, and ferritin levels. TTC staining evaluated infarction size. HE staining observed histopathological changes. DCFH-DA fluorescent probe detected ROS. CCK-8 kit measured cell viability. HiSeq 2000 sequencing performed differential expression analysis of miRNAs. qRT-PCR and Western blots assessed the expression levels of GPX-4, ACSL-4, HO-1, TFR-1 and TFR-2. SPSS 21.0 software conducted statistical analysis.
Results
Myocardial ischemia-reperfusion injury resulted in decreased levels of SOD and GSH, increased levels of LDH and MDA, up-regulation of ferritin, ACSL-4, HO-1, and TFR-2, down-regulation of GPX-4, increased tissue damage, and accumulation of ROS. However, DFO treatment reversed these changes. Subsequently, the target gene miRNA-541-5p was obtained by miRNA sequencing screening, and further validation revealed that miRNA-541-5p expression was increased in the myocardial tissues of rats in the I/R injury model group compared with those of rats in the NC group, P < 0.05. Subsequently, by constructing H9C2 cell lines with miRNA-541-5p overexpression and miRNA-541-5p expression inhibition, miRNA-541-5p expression was inversely correlated with the survival of H9C2 cells after hypoxia-reoxygenation injury. miRNA-541-5p up-regulation led to a decrease in SOD and GSH, an increase in ferritin and MDA, and an accumulation of ROS. wb and qRT-PCT demonstrated that high miRNA-541-5p expression up-regulated the expression of protein/mRNA expression of ACSL-4, HO-1, ferritin, and TFR-1, but down-regulated protein/mRNA expression of GPX-4. In addition, ADAM 7, FNIP 2, HOXD 10, HCCS and STK 3 were preliminarily identified as potential candidate target genes for miRNA-541-5p by bioinformatics analysis. Among them, ADAM7 emerges as the most suitable potential target gene based on the selection criteria.