| Background Micro RNAs(mi RNAs)are an abundant group of endogenous noncoding RNA molecules,which are highly conservative on evolution,approximately composed of 19-25 nucleotides.It combines with the m RNA 3' untranslated regions to inhibitive the protein translation process or promote degradation,thus negatively regulate the expression of target genes.Micro RNAs involve in a variety of regulation ways,including development,cell proliferation differentiation and apoptosis,organ formation,fat metabolism,etc,playing an important role in regulating gene expression in the growth and development of biology.The incidence and mortality of acute myocardial infarction(AMI)in China is high,and the onset of AMI has the trend of younger age.Several studies show that mi RNA plays an important role for in regulating cardiac angiogenesis and myocyte hypertrophy,fibrosis followed AMI.These findings clearly become established the heart ischemia diseases with the expression of mi RNAs,indicating the mi RNAs may become the target to improve recovery after AMI,especially acute inhibition or over expression after AMI may help reduce tissue damage,and improve the formation of new blood vessels,and prevent subsequent negative cardiac remodeling in heart failure.The research of mi RNAs in AMI offers a new way to solve the AMI.Through the establishment of rat model of AMI,we detect the mi RNA expression differences in myocardial tissue,so as to find the role of mi RNA in these pathways,to provide novel strategies for the clinical treatment of the patients with AMI.Purpose To establish the rats model of AMI and study the molecular mechanism of mi R-182 involved in apoptosis of AMI.Methods 1.Model establishment and validation: we chose 8 weeks male wistar rats,the rats were divided into two groups according to random number table method,namely,the experimental group and control group(sham).The left coronary artery has been ligated in the experimental group rats,and myocardial ischemia lasted for 3 or 6 hours respectively,sham group was without ligation.After model establishment,we observed the presence of ischemia after the heart soaking in the 2,3,5-triphenyltetrazolium chloride(TTC)solution firstly.Secondly,we detected the serum markers of myocardial infarction troponin and myoglobin after ligation 3h and 6 h in rats.Again we tested the change of the heart tissue with HE staining(Hematoxylin-eosin staining).Finally,we used TUNEL staining to detect apoptosis in rats myocardial infarction model.2.Quantitative Real-time polymerase chain reaction(q PCR): We took Trizol method to extract RNA in AMI 3 hours,6 hours,and sham groups of heart tissue,and then reversed transcriptase m RNA synthesis to c DNA,to detect the mi RNA q PCR m RNA level.Finally we analysised the values of the gene express with 2-??CT.3.Western Blotting(WB)and immunohistochemical assay: To determine the protein level targeted by mi R-182 in myocardial tissue,we used the method of WB,then detected the expression of target protein Bax and caspase3 in histological level of myocardial tissue.4.H9C2 cell hypoxia model and the expression of mi R-182: We established model of H9C2 cell with hypoxia 6 hours,validating through the activity of LDH,cell proliferation and caspase3/7,and then detected the expression of mi R-182 in hypoxia model.5.Karyoplasm separation: Myocardial tissue and H9C2 cells were carried out according to the kit manual operation to extract the constituent of cell proteins,further to test protein expression changes in apoptosis of AMI.Results 1.We successfully established the rat model of AMI.TTC staining in the rat experimental group and sham group,non-infarct area were red,with a white infarction area.2.Myoglobin and troponin increased significantly in experimental group.3.Myocardial infarction area was observed structure disorder after HE staining,and inflammatory cells infiltration.4.TUNEL results revealed that infarction area of the rat experimental group appeared obviously apoptosis.5.QPCR results showed that,compared with the sham group,the expression of mi R-206 after infarction 6 hours rised significantly,mi R-1b,mi R-10b-5p,mi R-122,mi R-132,mi R-133a-3p,mi R-155,mi R-182 expression decreased obviously.But mi R-182,mi R-155 were in a significant reduction after infarction 3 hours,mi R-10b-5p did not appear this kind of situation.6.WB tested candidate targets cleaved-PARP-1,Mecp2,Fox O3 a,Hif-1?,p53.Results showed that cleaved-PARP-1,Mecp2,Hif-1?,Fox O3 a expression in infarction group and sham group were without differences.To the contrary,the expression of p53 obviously up-regulated after infarction 6 h compared with the sham group.Downstream of p53 apoptosis related proteins Bax,cytochrome c,caspase3 also increased in the myocardial infarction area of infarction group obviously.WB tested cell components of protein,according to the results of p53 in myocardial infarction area increased significantly in the nucleus.7.In histological level we detected the expression of Bax and caspase3,finding that Bax and caspase3 infarction area expressed higher than sham group.8.With H9C2 cell hypoxia 6 h,LDH released increased,caspase3/7 activities raised,and cell activity reduced.QPCR detection of mi R-182 expression levels,the result showed that the expression of mi R-182 was lower in hypoxia group than normal one.To extract the constituent of the cell protein,WB results showed that the expression of p53 in hypoxia group increased significantly in the cell nucleus compared with normal group.Conclusions 1.Mi R-182 was significantly lower in rat AMI model and H9C2 hypoxia model.2.Mi R-182 may be involved in the process of the pathological changes of acute myocardial infarction in rats through p53-Bax signaling pathway regulating apoptosis. |
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