Effects And Mechanism Of MicroRNA-155 On Ventricular Remodeling And Cardiac Function After Acute Myocardiac Infarction

Acute myocardial infarction (AMI) is myocardial necrosis caused by aute and sustainable ischemia and hypoxia of conranry artery. After myocardial infarction, some factors such as neurohumoral regulation disorder, immune inflammation, myocardial ischemia and cardiac stress, lead to ventricular remodeling whose features include the disorders of myocardial cells and interstitial cell. How to prevent and treat ventricular remodeling after myocardial infarction has become a hot spot and challenge.MicroRNAs, a class of short (approximately 22 nt), noncod-ing RNAs, regulate gene expression at the posttranscriptional level by inhibiting messenger RNA (mRNA) translation. Upregulation of miR-155 is found in various inflammatory diseases, such as rheumatoid arthritis and atherosclerosis. The expression of miR-155 is significantly increased in the serum of patients with myocardial infarction, and is associated with caridiac death with one year after discharge. However, the role of miR-155 in ventricular remodeling post MI remains unclear. In the study, we investigate the effect of miR-155 on left ventricular function and ventricular remdoeling after MI and its underling mechanism in mouse model constructed by ligation of the anterior descending coronary artery.Part 1:Effects of miR-155 on cardiac function and ventricular remodeling after acute myocardial infarction in miceObjective:To explore effects of miR-155 on cardiac function and ventricular remodeling after myocardial infarctionMethods:The animal model of AMI was established via ligation of the left anterior descending coronary artery (LAD) in WT C57BL/6J mice, miR-155+/+ and miR-155-/- mice. Mice in sham-operated group underwent the same procedure, but without the ligation. After 1,7 and 14 days, mice were performed by transthoracic echocardiography detection before sacrificed.The changes of infarct size, left ventricular dilatation index and the thickness of the scar were analyzed by TTC staining and Masson staining. The expressions of BNP and cTnI in cardiomyoctye were measured by immunohistochemical staining.Results:(1) Compared to the miR-155+/+ and the WT group, cardiac function is obviously improved in the miR-1554 group. (2) Infarct size was significantly decreased in the miR-155-/- mice compared to the WT group at day 14 after LAD ligation. In contrast, the infarct size of the miR-155+/+ mice was significantly increased compared to that of the WT mice or miR-155-/- mice. (3) Mice in the miR-155-/- groups had a lower relative scar thickness compared with mice in the WT and miR-155+/+ group; In contrast, the scar percentage of whole LV circumference and LV expansion index were significantly lower in the miR-155-/- group than in the WT and miR-155+/+ group. (4) The expressions of BNP and cTnl were significantly inhibited in the miR-155-/- AMI group.Conclusion:miR-155 deteriorated cardiac function and ventricular remodeling after myocardial infarction.Part 2 Effects of miR-155 on the collagen synthesis, apoptosis, proliferation and differentiation in mouse cardiac fibroblastsObjective:Myocardial fibrosis contributes to the ventricular remodeling post-MI, leading to the development of chronic heart failure. Fibroblasts play a key role in myocardial fibrosis. The aim of this part was to explore the role of miR-155 in collagen synthesis, apoptosis, proliferation, and differentiation of mice cardiac fibroblast.Methods:The adult cardiac fibroblast and cardiomyocytes were prepared from WT, miR-155-/- and miR-155+/+ mice on day 14. The mRNA expressions of miR-155 and TP53INP1 were detected by RT-PCR. Primary cardiac fibroblasts were stimulated with TGF-β1 (5ng/mL) for 24,48, or 72 hours. The expression of miR-155, collagen I, collagenⅢand caspase-3 were detected by RT-PCR. The protein level of cleaved caspase-3 was detected by Westernblotting. The proliferation of neonatal mouse cardiac fibroblasts was measured by MTT assay. Immunofluorescence staining was performed to detect the expression of a-SMA.Results:(1) Compared with sham group, the expression of miR-155 in the cardiac fibroblasts was significantly increased in the WT and miR-155+/+ mice post MI. However, there is no difference in the cardiac myocytes post MI. In contrast, TP53INP1 expression in the cardiac fibroblasts was significantly down-regulated post-AMI, whereas there was no significant change in cardiac myocyte between the sham and AMI groups of WT mice. The expression of TP53INP1 in both the cardiac myocytes and fibroblasts was significantly reduced in the miR-155+/+mice, but was increased in the miR-155-/- mice. (2) TGF-β1 significantly increased the expression of miR-155 in WT cardiac fibroblasts in a time-dependent manner. mRNA expression of collagen Ⅰ/Ⅲ was dramatically up-regulated exposure to TGF-β1. The expression was also higher in the miR-155+/+ group than in the WT group; however, knock out of miR-155 abolished the TGF-β1-mediated enhancement of collagen Ⅰ/Ⅲ. (3) In the WT group, caspase-3 mRNA and cleaved caspase-3 protein expression was dramatically and time-dependently down-regulated by TGF-β1. The expression was also lower in the miR-155+/+ group than in the WT group. In contrast, miR-155 KO significantly reversed the TGF-β1-mediated down-regulation of caspase3. (4) Overexpression of miR-155 promoted cell proliferation and knock out of miR-155 significantly inhibited cardiac fibroblast proliferation.(5) The expression of a-SMA was significantly higher in the miR-155+/+　group than in the WT group, and lower in the miR-1557-/- group.Conclusion:The expression of miR-155 was significantly increased in fibroblasts post myocardial infarction. miR-155 increases the synthesis of collagen I /Ⅲ, inhibits the apoptosis of myofibroblast, and promotes the proliferation of murine fibroblast and differentiation to myofibroblast, which may be associated with down-regulation of TP53INP1.Part 3 Effects of miR-155 on the collagen synthesis, apoptosis, proliferation and differentiation in mouse cardiac fibroblasts by targeting TP53INP1Objective:To explore effects of miR-155 on collagen synthesis, apoptosis, proliferation and differentiation of cardiac fibroblast by targeting TP53INP1Methods:The binding site of miR-155 was predicted by two bioinformatics tools and was validated by a dual-luciferase reporter assay. The cardiac fibroblasts were transfected with miR-155 mimics or inhibitors. TP53INP1 mRNA and protein expression level was measured by RT-PCR or Westernblotting, respectively. The primary neonatal mouse cardiac fibroblasts from WT or miR155-/- mice were transfected with si-RNA mediated downregulation of TP53INP1. The expressions of collagen Ⅰ/Ⅲ and caspase-3 were analyzed by RT-PCR. The protein level of cleaved caspase-3 was detected by Westernblotting. The proliferation of neonatal mouse cardiac fibroblasts was measured by MTT assay. Immunofluorescence staining was performed to detect the expression of a-SMA.Results:(1) TP53INP1 was a potential target of miR-155 and was validated by a dual-luciferase reporter assay. miR-155 inhibited the expression of TP53INP1. (2) mRNA and protein expression levels of collagen Ⅰ/Ⅲ were dramatically upregulated in the si-TP53INP1 group, while the mRNA levels of caspase-3 and protein levels of cleaved caspase-3 were significantly downregulated.(3) si-TP53INP1 significantly promoted cell proliferation and increased the expression of a-SMA in the WT and the miR-155 KO cardiac fibroblasts compared to the si-NC fibroblasts.Conclusion:miR-155 deteriorated cardiac function and increased myocardial fibrosis after acute myocardial infarction by targeting TP53INP1 in fibroblasts...