MicroRNAs (miRNAs) are a class of highly conserved endogenous noncoding single-stranded small RNAs with a length of 18-22 nucleotides. They are involved in regulation at the posttranscriptional and translational levels through the degradation and translation inhibition of messenger RNA (mRNA). It is estimated that at least 60% of all mammalian genes may be regulated by miRNAs. MiRNAs can help uncover the mechanisms of diseases and provide new entry points into therapy. Accumulated evidence has revealed that miRNAs are involved in the pathological process of cardiovascular disease through specific signaling pathways. To investigate the potential miRNAs for myocardial fibrosis post myocardial infarction, rat models of acute myocardial infarction (AMI) were established by ligating the anterior descending branch of the left coronary artery, while sham-operated rats were only threaded without ligation as a control group. There were three rats in each group. Thirteen differentially expressed miRNAs between the two groups were screened and their expression levels in the model group were all higher than those in the control group. The expression of miR-199a-5p was significantly increased in the model group in qRT-PCR, which was consistent with the results of the gene chip. KEGG enrichment analysis showed that the target genes of miR-199a-5p were enriched in the insulin signaling pathway. Furthermore, dual-luciferase reporter assay indicated that miR-199a-5p could negatively regulate the expression of GSK-3β. After transfection, the expression of miR-199a-5p was increased in the miR-199a-5p mimics group. The protein expression of GSK-3β was decreased in CFs transfected with miR-199a-5p mimics. In summary, our study identified miR-199a-5p could promote the progression of myocardial fibrosis after myocardial infarction by targeting GSK-3β, which provides novel targets for diagnosis and treatment of MF. In summary, our research suggests that miR-199a-5p promotes the progression of myocardial fibrosis after myocardial infarction through the activation of the insulin-PI3K/Akt-GSK-3β signaling pathway.
Overall design: SPF male Sprague–Dawley (SD) rats weighing 200±20 g were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. Raised in the Barrier Animal Laboratory of Dongzhimen Hospital, Beijing University of Chinese Medicine, with a 12 h light/dark cycle and ad libitum food and water. All protocols and applications of animals in this study were approved by The Animal Care and Welfare Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (No: 16-25). The rats were divided into model group and sham-operated group by random number table. Before surgery, the rat was anesthetized intraperitoneally with 1% sodium pentobarbital (40 mg/kg). The skin was cut laterally at the third and fourth ribs of the left chest, approximately 1.5 cm in length. The musculature was bluntly separated, the rib was braced with a thoracotomy and the thymus was fixed upward to fully enlarge the surgical field, and the pericardium was carefully torn open to expose the ligation site of the rat heart. The anterior descending branch of the left coronary artery was ligated with a 5/0 line approximately 2 mm below the left atrial appendage and the arterial cone. The anterior myocardial wall was quickly ischemic and whitening, and the electrocardiogram (ECG) revealed saddle-back-like ST-segment elevations. After 24 h, the ECG was conducted again, and pathological Q waves appeared in the chest leads and lead I and aVL, indicating that the model was successful. The sham-operated rats were only threaded without ligation as a control group. The rats were fed adequate food and deionized water for 4 successive weeks, subsequently euthanized and dissected to isolate the heart for the subsequent experiments. Left ventricular samples were obtained for RNA extraction and hybridization on Agilent miRNA Microarrays.
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