The Relationship Between Plasma Circulating MicroRNAs And Major Adverse Cardiac Events After PCI

MicroRNA (miRNA) is an extensive class of endogenous non-coding small single strand RNA molecule which length is about21-25ribonucleotide. In1993, lee.et found the first miRNA named lin-4which can regulate the growth of nematode larvae by inhibiting expression of nucleoprotein Lin-14in nematode. Since then new miRNAs are always isolated and identified by scientist. There is more than2109hunman miRNAs has been found by April2012(http://www.mirbase.org). Along with the deepening of the study, an extensive class of new miRNAs will be found.Plasma circulating miRNAs may be a class of potential clinical novel biomarkers. Current results of miRNAs detected in healthy people and people with different disease indicates that miRNAs molecular was widely exists in hunman plasmajust like the circulating nucleic acid(DNA and RNA).Despite the function of plasma circulating miRNAs has not been clear yet, It’s have some good properties for the potential clinical novel biomarkers, just like the cell specificity, tissue specificity and the disease specificity, these specific expression is its function foundation and the good biological properties for the potential clinical novel biomarkers. More over, plasma circulating miRNAs were relatively stable display in recent study, which was an ideal of clinical novel biomarkers required. Therefore, profound study of abnormal expression of plasma circulating miRNAs under different disease states can contribute to the diagnosis and prognosis of diseases in clinical work.Coronary heart disease is one of the most important causes of death, and also consumes huge medical resources of disease. Percutaneous coronary intervention (PCI) is one of the significant progress in the treatment of coronary heart disease. With its precise and significant clinical curative effect, it can improve patients’ quality of life and survival by improving the symptoms of ischemia and hypoxia of patients.Due to the stents implantation in the PCI operation, it can provoke platelet activation, thrombosis formation, bracket within thrombosis, myocardial infarction death, and other clinical end event. As for clinical end event,10%-15%of patients will have heart and brain vascular event within1year, including brain stroke and major adverse cardiac events (MACE including heart death, myocardial infarction and reconstruction).Based on the new progress on plasma circulating miRNAs, we believe that the variety and expression level of plasma circulating miRNAs in patients was relatively stable, and can balance the state after PCI. Change of cardiac function in patients with adverse reaction, metabolic abnormality or organic disease, may directly or indirectly destroy the homeostasis of plasma circulating miRNAs in ways of changing the expression level of certain class plasma circulating miRNAs. Therefore, this research will establish a mature and reliable method of plasma circulating miRNAs extraction and detection, as well as explore the relationship between the expresstion levels of plasma circulating miRNAs and the prognosis of patients after PCI and look for clinical novel biomarkers to evaluate the prognosis of patients after PCI. Besides, after the differences expression level of circulating miRNAs has been found in the former clinical research, we further explore the miR-126biological function on vascular endothelial cells.Part Ⅰ Quantification of plasma circulating miRNAsAim: To establish a stable method for the extraction of plasma total RNA and quantification the expression level of circulating miRNAs in Plasma.Method: First blood specimens from healthy volunteers were collected and then separated plasma specimen by gradient centrifugation. In the aspect of extraction, Trizol LS kit was used to extract the plasma total RNA. And then further confirm there are small RNA molecule in the total RNA by the method of capillary electrophoresis using Agilent2100. In the aspect of quantification, a certain concentration gradient of nematode species cel-miR-39was added to inspect the specific property of extraction and detection methods. In the end, identifiation of specific property of the amplification product was done by agarose gel electrophoresis.Result: The concentrations of total RNA in plasma extracted from10healthy volunteers are between lng/μL and8ng/μL. While a gradient dosage of cel-miR-39as0pmol、2pmol、20pmol、200pmol、2000pmol was added, the quantification result from RT-qPCR showed that the Ct Value is>40、30.33、28.99、26.71、24.00respectively.By detecting the endogenous miR-16and miR-126expression in plasma using specific design primers, we found that miR-16is abundance and relatively stable while miR-126is relatively low expressed in plasma. The amplified products of miR-16, cel-miR-39and miR-126by RT-QPCR are specific, presenting a single line about70-80bp on the Agarose gel electrophoresis, which has adjust our primer design.Conclusion: There are abundant miRNAs exist in the plasma which can be extracted by the Trizol LS kit as the total RNA. The method of plasma circulating miRNAs quantification is reliable and effective.Part Ⅱ The relationship between plasma circulating MicroRNAs and major adverse cardiac events after PCIAim: The blood samples with patients after PCI were collected and then follow up in one year. The expression levels of candidate miRNAs (miR-126、miR-21、miR-26b、 miR-223、miR-16)were further measured in those patients,and then analyed the relationship between plasma circulating miRNAs levels and major adverse cardiac events after PCI,explore the risk factor of MACEMethod:Base on the quantification method of plasma circulating miRNAs established in part Ⅰ,480blood samples with patients after PCI were collected and the expression levels of candidate miRNAs (miR-126、miR-21、miR-26b、miR-223、 miR-16)were measured.In order to explore the relationship between plasma circulating miRNAs levels and major adverse cardiac events after PCI, we divided the expression miRNAs levels into three group [Ql:low expression level(25%); Q2:middle expression level(50%); Q3:high expression level(25%)]. Eventually, we explore the risk factor of MACE through the Cox regression model.Result:(1) There are480patients in our research.The main follow up result shown as below:4patients(0.83%) with thrombosis in coronary stents after percutaneous coronary intervention;24patients(5.00%) have major adverse cardiac events after percutaneous coronary intervention; The5candidate miRNAs (miR-126、miR-21、 miR-26b、miR-223、miR-16)can be detected in the plasma of the patients after percutaneous coronary intervention.Base lines in each miRNAs expression levels show no significant differences in the three groups.(2) By evaluation of the in-stent thrombosis as the end points, we found that the total cumulative survival rates have significant differences in the three group of miR-16expression levels(χ2=6.077, P=0.048)thought Log-rank single factor analysis. Comparing to the Q2levels, the total cumulative survival rates of Q3group has significant reduced(χ2=6.096, P=0.014); The total cumulative survival rates have no significant differences in the three group of miR-126, miR-21, miR-26b, miR-223expression levels (P>0.05).(3)By evaluation of the End joint events as the end points, we found that the total cumulative survival rates have significant differences in the three group of miR-126expression levels(χ2=6.154, P=0.046)thought Log-rank single factor analysis. Comparing to the Q1levels, the total cumulative survival rates of Q3group has significant reduced(χ2=6.460, P=0.011); The total cumulative survival rates have significant differences in the three group of miR-223expression levels(χ2=8.218, P=0.016), Comparing to the Q1levels, the total cumulative survival rates of Q3group has significant reduced(χ2=6.450, P=0.011); Comparing to the Q2levels, the total cumulative survival rates of Q3group has significant reduced(χ2=4.436, P=0.035). The total cumulative survival rates have no significant differences in the three group of miR-21,miR-26b and miR-16expression levels(P>0.05). (4) The result of the Cox regression model present that the miR-21expression levels、 miR-223expression levels,hyperpiesia,Left ventricular ejection fraction and low density lipoprotein are in the regression model, which shows high miR-233, hyperpiesia,low express of low density lipoprotein and miR-21,the reduce of Left ventricular ejection fraction are risk factor with major adverse cardiac events after PCI.Conclusion: The five candidate miRNAs (miR-126、miR-21、miR-26b、miR-223、 miR-16) are all express in the patients after PCI. With the high expression levels of miR-126and miR-223,the cumulative survival rate of patients is significant low compare to the other two expression levels.Plasma circulating miR-126and miR-223may be candidate clinical novel biomarkers for the prognosis of the patients after PCI.Part Ⅲ The effect of miR-126on vascular endothelial cellsAim:Explore the effects of miR-126on vascular endothelial cell.Method:Eahy-926cells line was cultured in vitro and then transfected with miR-126mimics and miR-126inhibitor by cationic-mediated transfection methods.36hours after transfected miR-126mimics and miR-126inhibitor, total RNA was extracted from culture cells, and mRNA expression of VEGF was detected by fluorescent quantitative PCR. Meanwhile, total protein was extracted by cracking cell and then the expression level of the VEGF protein was detected.Result: Our result indicates that miR-126have regulated the VEGF expression by inhibitor miR-126and promote miR-126expression in cell lines.36hours after transfection of miR-126inhibitor50nM, the VEGF mRNA and protein have significantly changed in the three group(F=46.128,P<0.001; F=44.959,P<0.001), the VEGF mRNA and protein have significantly promoted comparing to the negative control(P<0.001,P<0.001).36hours after transfection of miR-126mimics50nM, the VEGF mRNA and protein have significantly changed in the three group(F=96.542, P<0.001; F=15.425,P=0.004),the VEGF mRNA and protein have significant decreased comparing to the negative control(P<0.001,P=0.003). Conclusion: Reduction of VEGF protein expression level may due to the binding of miR-126to the3’ untranslated region of the VEGF mRNA. On the other hand, VEGF protein can be promoted when miR-126was inhibited in Eahy-926. miR-126have negative regulation on the expression of VEGF in Eahy-926cell lines.