Role Of MicroRNA-26 In Rat Cardiac Remodeling

[Backgroud] Cardiac remodeling is associated with heart injury or hemodynamic stress response, and then induces the changes of heart structure and function due to the variations of gene expression and protein regulation. Continuous cardiac remodeling will ultimately lead to heart failure (HF) which is the important reason for clinically patients with arrhythmias and sudden death. The methods to reverse cardiac remodeling was still deficiency in nowadays, people in the previous researches always focused on the post-translational regulation of neurohumor and signal transduction pathway in the pathogenesis of cardiac remodeling, while the research of the mechanisms of post-transcriptional control was not so much. MicroRNA are a series of single-stranded non-coding RNA molecular with the length of 19~25 nt, they achieve post-transcriptional negative regulation through combination with the target gene to inhibit or degrade mRNA. In previous studies, it was shown that many miRNA expression levels changed accompanying with the process of cardiac remodeling. Among these studies, the miRNA expression microarray indicated that the expression of miR-26 was down-regulated in the myocardium of the cardiac remodeling animal models. Therefore, we supposed that up or downregulation of miR-26 may cause variation of the phenotype of cardiac myocytes. GSK3βwas demonstrated playing an important role in the signal transduction pathway in the development of cardiac remodeling/heart failure, and it contain the predicted binding site of miR-26. Therefore, we assumed that miR-26 function in the cardiac remodeling may act through regulating the expression of GSK3β. In this study, we established rat models of cardiac remodeling and cell models to investigate whether miR-26 is involved in cardiac remodeling, cardiac hypertrophy and fibrosis. Dual luciferase reporter system was used to validate the target site of miR-26 on the GSK3β3'-UTR.[Purpose]1. To illuminate the role of miR-26 in the rat cardiac remodeling models and cell models.2. To elucidate the relationship among miR-26, GSK3βand cardiac remodeling on rat cardiomyocytes (CM) and cardiac fibroblasts (CF) in vitro.[Methods]1. Used Transverse Abdominal Aortic Constriction (TAAC) to establish rat models of overload pressure cardiac remodeling. The changes of heart's structure and function were detected with animal echocardiography, the mRNA and protein expression of hypertrophy gene were detected with QPCR and western blot, the cell area and percentage of fibrosis were detected with histopathology staining methods, all the results indicated that the model was successful.2. QPCR assay was applied to detect the expression levels of miR-26 in the myocardial tissue and plasma in TAAC rats.3. Primary CM and CF from neonatal rat were Cultivated in vitro, and QPCR assay was used to detect the expression levels of miR-26 in CM and CF after being stimulated by normal culture and AngiotensinⅡ.4. CM and CF were transfected with miR-26 mimics and inhibitor, and miR-26 expression level was tested by QPCR assay.5. CM and CF were transfected with miR-26 mimics, and then induced by AngiotensinⅡ, or CM and CF were transfected with miR-26 inhibitor alone. western blot and QPCR assay were used to detect the mRNA and protein expression of hypertrophy gene and collagen, tritium labeled leucine incorporation was employed to detect the rate of protein synthesis in CM, and immune cell morphological was applied to detect of CM morphology.6. Western blot and QPCR assay were used to detect the level of GSK3βexpression, phosphorylation, and the GSK3βactivity in the TAAC rat myocardial tissue and the AngiotensinⅡstimulated-CM and CF.7. Used western blot and QPCR assay to detect the levels of GSK3βand its phosphorylation, and the activity of GSK3βwhile transfecting CM and CF with miR-26 mimics and inhibitor.8. Dual luciferase reporter system was applied to validate whether GSK3βis the target gene of miR-26.9. CM and CF were transfected with small molecule RNA to interfere GSK3β, and then induced by AngiotensinⅡ. QPCR assay were used to detect the mRNA expression of GSK3β, hypertrophy gene and collagen.10. CM and CF were transfected with plasmid to overexpress GSK3β, western blot and QPCR assay were used to detect the expression of GSK3β, hypertrophy gene and collagen.[Results]1. Rats model of overload pressure cardiac remodeling was established successfully by the method of TAAC.2. MiR-26a and miR-26b expression level in the myocardial tissue and plasma in TAAC rats all decreased compared with the sham group (P﹤0.05).3. After stimulating CM and CF with AngiotensinⅡ, miR-26a and miR-26b expression level all decreased comparing with the control group (P﹤0.05).4. MiR-26 expression level were much higher in normal cultured CM than that in CF (P﹤0.05).5. After transfecting CM and CF with miR-26 mimics, miR-26a and miR-26b expression level all increased significantly (P﹤0.01); while transfecting the two cells with miR-26 inhibitor, and the expression level of both miR-26a and miR-26b decreased (P﹤0.05).6. MiR-26 mimics inhibited the effect of hypertrophy or collagen synthesis induced by AngiotensinⅡ, and the hypertrophy gene or collagen expression decreased (P ﹤0.05), the rate of protein synthesis slowed down (P﹤0.05), and cell area shrinked (P﹤0.05).7. After transfecting CM or CF with miR-26 inhibitor, hypertrophy or collagen gene expression increased (P﹤0.05),α-actin or Collagen 3 level increased, the rate of protein synthesis accelerated in CM (P﹤0.01), and CM cell area augmented (P﹤0.01).8. The mRNA and protein levels of GSK3βall increased (P﹤0.05) in the TAAC rat myocardial tissue, and the strenghthened levels were accompanied with its activity, while the expression of the phosphorylation GSK3βdid not chang significantly.9. After stimulating with AngiotensinⅡ, the mRNA levels of GSK3βall increased in CM and CF (P﹤0.05).10. The mRNA and protein level of GSK3βall decreased (P﹤0.05) while transfecting CM or CF with miR-26 mimics, and increased (P﹤0.05) while transfecting with miR-26 inhibitors.11. After transfecting CM and CF with miR-26 mimics, GSK3β3'UTR report gene vector luciferase expression were inhibited and then luciferase activity followed to descend (P﹤0.01); while transfecting them with miR-26 mimics, GSK3β3'UTR report gene vector luciferase expressions augmented and then luciferase activity followed to enhance (P﹤0.05); all of these ensured that GSK3βis the target gene of miR-26.12. SiGSK3βinhibited the effect of hypertrophy or collagen synthesis induced by AngiotensinⅡin CM or CF with the hypertrophy or COL3 gene decreased (P﹤0.05).13. After using plasmid to overexpress GSK3βin CM or CF, both the expression level and activity of GSK3βsignificantly increased (P﹤0.05), meanwhile the levels of hypertrophy or Collagen gene augmented (P﹤0.05). [Conclusions]1. MiR-26 is down-regulated in rat cardiac remodeling model.2. MiR-26 regulates the process of cardiac remodeling possibly through inhibiting the expression of GSK3β.