Expression Profiling Of MiRNA In Patients With Unstable Angina

Unstable Angina (UA) is a serious type of coronary atherosclerotic heart disease,which is a clinical syndrome between chronic stable angina and acute myocardial infarction. Unstable angina is a precursor of AMI.The occurrence of acute myocardial infarction (AMI) is 12%-13% with 3-18% of mortality rate in unstable angina (UA) patients within one year. Currently, the diagnosis of UA is based on clinical symptoms, ECG and coronary angiography. We need a sensitive biochemical indicators for early diagnosis.Therefore, Looking for the early diagnosis of molecular markers, has become the hot topic of cardiovascular field.MicroRNA is a class of noncoding single-stranded RNA molecules which is about 20-24 nucleotides in length. MicroRNA has been confirmed to participate in the development of cardiovascular, which plays an important role in the development of atherosclerotic disease.MiRNA can be stable present in the circulatory system in the form of microbubbles, protein complexes. This feature gives the molecular diagnosis of cardiovascular disease provide the material foundation. Currently, chip technology and qRT-PCR has been widely used in plasma miRNA screening and validation of differentially expressed genes. This study was used Exiqon LNA miRNA chip technology and TaqMAN stem-loop miRNA quantitative PCR, comparative analysis the plasma miRNA of expression profiles between UA patients and healthy, Screening differentially and stably expressed plasma miRNA molecule,then used quantitative PCR to validate it.The technical routes in this study:firstly,we collected plasma samples from UA patients and healthy individuals, those samples were used for plasma miRNA microarray and qRT-PCR validation.Secondly, we collected plasma samples again from healthy individuals, analyzed the expression of RNU6B in plasma and cell,and processed the chip data with RNU6B expression level What shall prevail, Obtained high reliability molecules of differentially expressed and internal control.Third, verification by quantitative analysis, we will find the expression of candidate genes miR-9-3p in the plasma whether there are significant differences in UA patients and healthy people. we explore the role of miR-9-3p in regulating ABCA1 expression which is to be a crucial molecular in cholesterol metabolism in vitro. Provide evidence for miR-9-3p involved in UA patients’ lipid regulating, in order to provide potential molecular markers for the early diagnosis of UA.PART 1 Expression profiling of plasma miRNA in unstable angina patientsObjective:To screen differential miRNA profiling in plasma from unstable angina patients.Methods:The research enrolled 175 plasma samples from healthy subjects and 150 plasma samples from UA patients, to establish 7 plasma pools of healthy subjects and 6 pools of UA patients. MiRCURY LNATM method is applied to detect expression profiling of plasma miRNA. Using statistical analysis software R 3.0.2 in limma microarray analysis program package to finish miRNA microarray data analysi.Results:1 Basic characteristics of these volunteers in this studyHealthy subjects (n=175) including 109 male and 66 female,UA patients (n=150) including 84 males and 66 females,on an average age of 54±11,55±9 years,were selected. Clinical data included age,sex,FPG,TC,TG, HDL-C,and LDL-C,all data were not statistically significant between healthy group and UA group (P>0.05) (Table 1).2 Plasma miRNA microarray data acquisition and analysis2.1 Normalized the plasma miRNA microarray dataChip data normalized to eliminate the overall bias of the fluorescence signal. Before VSN normalization, the standard deviation of the mean is rising; after VSN normalization, inter-chip signal overall bias is eliminating,as shown in Fig.1,Fig.2.2.2 Correlation analysis of plasma miRNA microarray dataSpearman correlation analysis showed that the plasma miRNA expression profiling between the healthy control group and UA group was repeatable,the correlation coefficient was 0.87 ± 0.08 (Table 2) and 0.94±0.05 (Table 3).2.3 The volcano plot of plasma miRNA microarray dataVolcano plot showed that there are a lot of different miRNA molecule expression between the two groups in the plasma (Fig.3 upper left and upper right quadrant quadrant)2.4 Differential expression analysis of plasma miRNA in UA group207 miRNAs are up-regulated and 134 miRNAs are down-regulated in UA patients when p value less than 0.01 were considered as a cut-off level.Conclusion:1 Use Exiqon miRNA chip technology, we got the expression profiling of plasma miRNA from healthy control group and UA group.PART 2 The plasma miRNA expression profiling of UA patients after adjustment of plasma RNU6B baselineObjective:To obtain high reliability of UA plasma miRNA expression profile of patients by adjustment of plasma RNU6B baseline.Methods:The research selected 10 cases of healthy volunteers (5 males, 5 females) for this study, TaqMan probe and qRT-PCR are applied to detected the plasma expression level of RNU6B,miR-423-5p and miR191-5p.Experimental data were analyzed using 2-ΔCt, two independent samples t test or Mann-Whitney U test, Data were analyzed using SPSS 16.0 software. Data were plotted using GraphPAD Prism 5.0 software. P<0.05 was considered as statistical significance.Results:1 Basic characteristics of these volunteers in this studyThe average age of subjects was 43± 6 years old, male to female ratio of 1:1,The FPG is 5.15±0.56 mmoL/L,The TC is 4.95±0.77mmoL/L,The TG is 1.13±0.35 mmoL/L,The HDL-C is 1.04±0.32 mmoL/L,The LDL-C is 2.94±0.81 mmoL/L (Table 4).2 qRT-PCR are applied to detected the 10 healthy volunteers plasma expression level of RNU6B,miR-423-5p and miR191-5p.2.1 Lateral comparison of the expression levels of RNU6BRNU6B plasma expression levels were significantly lower than the level of expression in HepG2 cells RNU6B (1.26 10-11 vs 2.04 10-8;P< 0.001) (Table 5, Fig.4).2.2 Analyze the expression level of RNU6B and miR-423-5p in plasmaRNU6B plasma expression levels were significantly lower than the expression level of miR-423-5p plasma.(1.26 10-11 vs 7.94 10-8;P< 0.001) (Table 6,Fig.4)2.3 Analyze the expression level of RNU6B and miR-191-5p in plasmaRNU6B plasma expression levels were significantly lower than the expression level of miR-191-5p plasma.(1.26 10-11 vs 1.03 10-8;P< 0.001) (Table 6, Fig.4)2.4 The plasma miR-508-3p and miR-509-5p qRT-PCR amplification cyc les are higher than the 40 Ct, beyond the qRT-PCR detection range, sli ghtly lower than the number of amplification cycles RNU6B’s 35-40 Ct.3 With plasma RNU6B baseline adjustment derived the plasma miRNA expression profilingUA group and healthy controls expression profiles of plasma charac teristics change significantly after adjustment of plasma RNU6B baseline, the difference was statistically significant (P<0.001;Table7,Fig.5).Ther e are 207 miRNA expression levels of molecules increase,134 miRNA expression levels of molecules decrease before adjustment.After adjustme nt, the upregulated miRNA molecules reduce to 124 (Table 8), the do wnregulated miRNA molecules reduce to 88 (Table 9)After adjustment between the two groups, stable miRNA variability was reduced from 938 to 355, suggested that without baseline data to adjust the chip contains a lot of no biological significance of the signal. Table 10 showed that there are 30 relatively stable miRNA molecules which is high levels of expression, including has-miR-423-5p which being recommended as a internal reference use.Conclusion:1 RNU6B plasma levels were significantly lower than the expression level of the cell.2 After RNU6B baseline adjustment, the expression profile characteristics of UA group and healthy controls significantly changed, after miRNA Microarray data analysis provided an experimental basis.Provide an experimental basis for data processing of miRNA microarray.PART 3 Analysis the levels of plasma miR-9-3p expression in UA patients and the effect of the expression levels of ABCA1Objective:To validate differential changes of miR-9-3p in plasma from UA patients. To explore the role of miR-9-3p in regulating ABCA1 expression which is to be a crucial molecular in cholesterol metabolism in vitro.Methods:The volunteers including 30 UA patients(15 males,15 females), 28 healthy controls(14 males,14 females). MirVana miRNA and TaqMan probe are applied to isolate and purify plasma RNA. The expression level of miR-9-3p is detected by qRT-PCR. Receiver-operating curve (ROC) analysis was used to evaluate its diagnostic significance for UA patients. In vitro experiments were performed using the techniques of cell culture, transiently transfection, qRT-PCR and western blotting to explore the role of miR-9-3p in regulating ABCA1 expression which is to be a crucial molecular in cholesterol metabolism in vitro.Data expressed as median (interquartile range). Relative levels of miRNA were reported as 2-ΔCt method. Fold change at transcriptional levels were reported as 2-ΔΔct method. Independent students’t test or non-parameters t test were used as appropriated. P<0.05 was considered as statistical significance. SPSS16.0 and GraphPAD Prism 5.0 software were used to analyzed data and drawing.Results:1 Basic characteristics of these volunteers in this study1.1 Healthy subjects (n=28) including 14 male and 14 female,UA patients (n = 30) including 15 males and 15 females, on an average age of 56±8,56±10 years,were selected. And the subjects from part 1 were excluded(Table 11).1.2 Clinical data including age,sex,FPG,TC,TG, HDL-C, and LDL-C were not statistically significant between healthy group and UA group (P>0.05) (Table 11).2 Compared with healthy group the plasma levels of miR-9-3p in UA group were significantly decreased (P<0.001,Table 12,Fig.6).The ROC curves of miR-9-3p with an area under curve (AUC) of 0.839 (95%CI:0.720 to 0.958) showed that plasma miR-9-3p can be accurate in diagnosing UA (P<0.0001,Fig.7).3 miR-9-3p influence ABCA1 protein expression in HepG2 cell3.1 The transiently transfected efficiency of miR-9-3p into HepG2 cell line were detected using qRT-PCR.The qRT-PCR result showed that the expression levels of miR-9-3p were significantly elevated 22.58 times higher than control group(P<0.001;Fig.8).3.2 qRT-PCR result indicated that there is no difference in ABCA1 mRNA levels between miR-9-3p tranfected group and control group (Fig.9,P=0.583).3.3Western blotting showed that protein levels of ABCA1 are significantly lower than control group(Fig.10,Fig.11,P=0.03).Conclusion:1 Patients with unstable angina,the expression levels of plasma miR-9-3p significantly decreased, plasma miR-9-3p may become one of UA diagnostic molecular markers.2 miR-9-3p inhibits the expression of ABCA1 protein at post-transc riptional level.