The Efficacy And Mechanism Of Microrna Abnormal Expressed Of CD4+T Lymphocytes In Unstable Angina Pectoris And Myocardial Injury After Percutaneous Coronary Intervention

Atherosclerosis is a chronic inflammatory response and autoimmune diseases, and the main pathological basis of coronary heart disease.The previous studies have shown that a large number of T lymphocytes can be found in atherosclerotic plaques, which mainly CD4+T lymphocytes. The imbalance of CD4+T cell subsets closely related with acute coronary syndrome. A large of number activated T lymphocytes in vulnerable plaque resulted in the reduction of plaque stability, which leads to plaque rupture easily. In patients with unstable angina and acute myocardial infarction, platelet thrombus memory has been found in myocardial microvascular and the formation of microembolism in the myocardial microcirculation in, leading to myocardial microinfarction. Thus, The CD4+T lymphocytes may be indirectly involved in the process of myocardial injury in patients with acute coronary syndrome.Myocardial injury after percutaneous coronary intervention remains a common complication, but its mechanism is not fully understood. Some studies have shown that preoperative dose of statins can effectively reduce the levels of inflammatory factors and protect myocardium. Research also shows that, The injury of the vessel wall related to the operation can lead to significant activation of peripheral blood lymphocytes and products of oxidative stress markers was significantly higher.The expression of CD18 in activated CD4+T cells of peripheral blood can promote the recruitment of leukocytes and adhere to the damaged endothelial cells. Therefore, the inflammatory response and the CD4 + T cell-mediated immune response may be directly involved in the process of myocardial injury after percutaneous coronary intervention.MicroRNA is a class of the small molecule RNA about 18 to 25 nucleotides. The recent studies confirm that miRNA are also involved in the regulation of monocytes/macrophages, lymphocytes and endothelial cells, which was closely related with development of atherosclerosis. Although the regulation of miRNA in T lymphocytes has made significant progress, but is still in its infancy.In different disease states and different stages of disease development, the miRNA expression and function varies. The function of miRNA in the regulation of T cell activation in the UAP and myocardial injury after PCI in is still unknown.In the present studies, the miRNA gene chip technology was adopted to screen abnormal expression of the miRNA in CD4+T lymphocytesh in patients with UAP. To observe the effect of abnormal miRNA expression on the CD4+T cell activation, differentiation. The changes of miRNA after PCI and the correlation with myocardial injury were also explored.Objective: To screen differential microRNA (miRNA) expression profiles of CD4+T lymphocyte from the patients with unstable angina pectoris (UAP) and the healthy controls by microarray analysis technique. To elucidate the mechanism responsible for modulation of CD4+T lymphocyte and provide insights into the effects of miRNA on UAP.Methods: Three patients with UAP were enrolled in the study, and three patients with normal coronary artery angiogram were served as a control group. Blood samples were taken from peripheral vein and the CD4+T lymphocytes were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cell sorting system (MACS). The purity of CD4+ T lymphocytes was measured by flow cytometry analysis. The viable count was detected by the rejection experiment of trypanblau. Total RNA was abstracted from CD4+T lymphocyte with Trizol reagent. MiRNA was isolated and enriched of by use of Polyethylene Glycol from 40μg total RNA. The miRNA extracted from CD4+T lymphocytes was hybridized and miRNA expressions profiles of CD4+T lymphocyte were screened with the Affymetrix GeneChip miRNA array. The image signal was scanned by Affymetrix GeneChip Scanner 3000 and analysised by Affymetrix GeneChip Command Console? 1.1 software. Then the image signal was transformed into digital information, which was analysised with SAM software. The differentially expressed miRNA were identified between the two groups. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to confirm the result of selected genes from microarray analysis.Results: The results showed that the expression of miR-155, miR-21, miR-424 and miR-127-3p were over 1.5 folds up-regulated, and the expression of miR-30b and miR-181a were over 0.5 folds down-regulated in UAP group compared to the control group. The qRT-PCR results were in accordance with those obtained using microarray analysis.Conclusion: The differentially expressed miRNA of CD4+T lymphocyte may participate in the the occuring and developing of UAP.KEY WORDS: Unstable angina pectoris;Lymphocyte;microRNA;Gene chip;Real-time polymerase chain reactionObjective: To investigate the effect of abnormal miRNA-155 expression on the differentiations and functions CD4+T lymphocyte in patients with unstable angina pectoris.Methods: Ten patients with UAP were enrolled in the study. Blood samples were taken from peripheral vein. The CD4+T lymphocyte were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cell sorting system (MACS). The CD4+T cells (2×106 cells/ml) were seeded in culture plates of 6 wells. Each well contained 2ml RPMI-1640 medium without 10% fetal bovine serum (FBS). There are four group CD4+T lymphocytes in the experiment: control group (transfected with 50nM N.C using Lipofectamine 2000), miRNA-155 group (transfected with 50 nM miRNA-155 using Lipofectamine 2000), miRNA-155 inhibitor group (transfected with 50 nM miRNA-155 and miRNA-155 inhibitor using Lipofectamine 2000), and FAM-siRNA group (transfected with FAM-siRNA). After stimulated with phytohemagglutinin, the CD4+T lymphocytes and culture supernatant were collected for the following experiments. The frequencies of Th1 and Th2 cells were measured by flow cytometry analysis (FACS). The total RNA and protein were extracted from CD4+T lymphocytes using Trizol and cell lysis buffer for western blotting, respectively. The level of IFN-γRα,T-bet,GATA-3 mRNA expression were measured by qRT-PCR. The level of IFN-γRα,T-bet,GATA-3 protein expression were examined using western blotting. The productions of IFN-γand IL-4 in culture superatants of CD4+T lymphocytes were detected by enzyme-linked immunosorbent assay (ELISA). Pearson correlation analysis was conducted to examine the association between IFN-γRαand IFN-γ, IL-4.Results: The FACS showed that miRNA-155 could promote CD4+T lymphocytes to Th1 cells and the Th1 frequencies were also significantly increased in miRNA-155 group compared with the control group and miRNA-155 inhibitor group[(63.19±8.61)% vs (47.17±10.28)%,P<0.01;(63.19±8.61)% vs (52.87±10.05)%,P=0.024,respectively]. There was no significant difference between the three groups in the frequencies of Th2 cells (F=0.228,P=0.798). In comparison with the control group, there was significant increase in the level of T-bet mRNA expression in miRNA-155 group (P<0.01). The level of T-bet mRNA expression in miRNA-155 inhibitor group was significantly lower than that in miRNA-155 group (P<0.01). No significant differences were found between the three groups in the level of IFN-γRαand GATA-3 mRNA expression (F=1.055, P=0.362; F=1.601, P=0.220, respectively). The level of IFN-γRαprotein expression in miRNA-155 group was significantly higher than that in miRNA-155 group and miRNA-155 inhibitor group (all P<0.01). There was no significant difference between the three groups in the level of GATA-3 protein expression(F=0.098, P=0.907).The culture supernatant concentration of IFN-γin miRNA-155 group was significantly increased than that in the control group and miRNA-155 inhibitor group (all P<0.01). No significant difference was observed between the three groups in the culture supernatant concentration of IL-4(F=0.384,P=0.685). There was significant negatibe correlation between the protein expression of IFN-γRαand the level of IFN-γ. No significant correlation were found between IFN-γRαand IL-4.Conclusion: It is evident that miRNA-155 can promote Th2 cells differentiations, and thus improving the function, which partly attributed to inhibit the expression of IFN-γRαprotein and may play an important role in the pathogenesis of unstable angina.Objective: To study the relationship of abnormal expressed miRNA-155 of CD4+T lymphocyte and myocardial injury after percutaneous coronary intervention in patients with unstable angina. To provide insights into the effects of miRNA-155 on myocardial injury in patients undergoing PCI.Methods: A total of 41 individuals with UA, who presented to the cardiology department of the First Affiliated Hospital of Guangxi Medical University were enrolled from January 2010 to December 2010. The patients were divided into two groups based on their troponin I (TnI) levels; elevated TnI group (TnI>0.15ng/ml, 20 cases) and normal TnI group (TnI≤0.15ng/ml, 21cases). Eighteen normal control subjects who were confirmed by coronary angiography were enrolled in the study. The level of miRNA-155 expression was measured by qRT-PCR before PCI and 12h after PCI. The level of IFN-γRαprotein expression were examined using western blotting and the level of serum IFN-γand IL-4 were detected by ELISA before PCI and 12h after PCI. Correlation analysis was made between miRNA-155 and the level of IFN-γRαprotein,serum IFN-γand IL-4.Results: Results: Compared with the control group, the expression of miRNA-155 and IFN-γwere significantly increased (all P<0.01), and the level of IFN-γRαwas markly decreased in the elevated TnI group and the normal TnI group before PCI (all P<0.01). There was no significant different in the level of IL-4 between the three groups before PCI (P>0.05). There was no significant different in the expression of miRNA-155,IFN-γRαand IFN-γbetween the elevated TnI group and the normal TnI group before PCI (all P>0.01). The expressions of miRNA-155, hsCRP, IFN-γwere significant increased, and the level of IFN-γRαprotein was decreased in the elevated TnI group and the normal TnI group at 12h after PCI (all P<0.05). The changes were more obvious in the elevated TnI group than the normal TnI group (all P<0.05). There was no significant different in the level of IL-4 in the elevated TnI group and the normal TnI group before and after PCI in. The expressions of miRNA-155 were significantly positive with IFN-γ(r=0.649 , P<0.01 ; r=0.682 , P<0.01, respectively), and significantly negative with IFN-γRαbefore and after PCI(r=-0.536 , P<0.01 ; r=-0.592 , P<0.01, respectively). There was no correlation between miRNA-155 and IL-4 before and after PCI (r=-0.165, P=0.303;r=0.107,P=0.506, respectively).Conclusion: The evidence showed that abnormal expressed miRNA-155 of CD4+T lymphocyte participated in the regulation process of Th2 cells differentiations, and function. There was correlation between abnormal expressed miRNA-155 and myocardial injury after PCI. MiRNA-155 may involve in the immunoregulation process of myocardial injury after PCI.