| Background:Ischemic heart disease(IHD), it can be also called coronary heart disease(CHD), becomes the most major cause of death. Studies have demonstrated that coronary heart disease is more of an inflammatory disease, which is affected by environmental and genetic interactions. Inflammatory response, hemodynamic changes, infection, stress, inflammation and other factors can aggravate the course of the disease. Because of the risk factors and pathogenesis of coronary heart disease are relatively complex, and racial differences, and genetic heterogeneity and other factors, therefore, pathogenesis of the disease is not very clear. Early diagnosis and early prevention is critical as in CHD, which prevents the development of disease from progressing, so that cure rate and survival improved.Studies have found that there are many miRNAs in peripheral circulation of blood, and that vascular endothelial-specific miRNAs are closely associated with diseases such as atherosclerosis and coronary heart disease. miRNAs are a class of highly conserved endogenous small-molecule non-coding single-stranded RNA, containing about 20 to 24 oligonucleotides, either sequential or tissue-specific is very strict, therefore, highly conservative in the evolution of species, and exists in almost all eukaryotic microorganisms. Mi RNAs usually have one or more target mRNA. Mi RNAs complementarily bind with the 3 ’end untranslated region(3’-UTR) of a specific target gene, which causes the degradation or translation inhibition, resulting in post-transcriptional regulation of gene expression. The study found that the outer peripheral circulating blood would have the presence of miRNAs, which, as a regulatory factor, plays an important role in both blood vessel development and angiogenesis. Up to now, about 800 miRNAs have been found in the human genome, and scientists have found that there are more than 1000 miRNAs capable of expression in the human genome. The study also showed that miRNAs might play a very important role in the treatment and prevention of heart disease and it could be a new therapeutic target for the treatment of coronary heart disease. It was reported that many miRNAs related to coronary heart disease. Among all the miRNAs, mi R-1, mi R-126,mi R-133, mi R-208 and mi R-499 have drawn much attention, and these vascular endothelial-specific miRNAs could be potential indexs for the patients with coronary heart disease. However, the evidence is not conclusive. Furthermore, the relation between these miRNAs expression levels in outer periphery circulating blood and the degree of coronary artery stenosis is unclear.Therefore, this study aimed to investigate the expression levels of mi R-133 a, mi R-208 a and mi R-499 a in peripheral blood in patients with coronary heart disease and their relation with the degree of coronary artery stenosis. Furthermore, we explore the value of Mi RNAs analysis in early diagnosis and prevention of coronary heart disease. Our study provides important theoretical basis for further study of gene diagnosis and treatment of coronary heart disease. Aims:1. Analysis of the differences from mi RNA-133 a, mi RNA-208 a and mi RNA-499 a expression level in patients with CHD and the control group.2. Analysis of correlations of mi RNA-133 a, mi RNA-208 a, mi RNA-499 a level and the degree of coronary artery stenosis, and exploring the clinical application value of miRNAs expression level in the early diagnosis and prevention of coronary heart disease. Methods:1. The study has been approved by ethics committee of the first affiliated hospital of the fourth military medical university. After obtaining consent from the patients and family members, we collected 290 CHD patients diagnosed with coronary artery angiography(CAG)from December 2014 to May 2015 in the department of cardiovascular medicine, the first affiliated hospital of the fourth military medical university. Age of these patients ranged from 30 to 85. Control group: 110 cases of hospitalized patients of the similar age, all excluded CHD diagnosis by CAG. CHD group: at least one of the three major coronary arteries presented more than 50% stenosis. Control group: none of the three major coronary arteries presented over 50% stenosis. Complete medical history and clinical data of all enrolled patients were recorded.2. Gensini score was a classic scoring method to evaluate the severity of coronary artery lesions. According to Gensini score, 400 cases were divided into four groups: 99 cases in A group: ≤ 22 points, 98 cases in group B: 23-55 points, 102 cases in group C: 56-96 points, 101 cases in D group, ≥ 97 points.3. 5ml of radial artery or femoral artery blood was extracted from all cases into blood collection tubes containing EDTA anticoagulant before coronary artery angiography. After two centrifugation, supernatant was dispensed into EP tubes and stored at-80 ℃ refrigerator. Coronary artery angiography was performed via radial or femoral artery.4. Determination of mi RNA expression levels: 500μl plasma was transfered to a new centrifuge tube after centrifugation. According to the protocol of the kit, all RNAs were treated with reverse transcription(RT)and stored in-80℃ refrigerator. c DNA was obtained from 20 ng total RNAs of each sample using PrimeScriptTM RT Master Mix(TaKaRa, Code No.RR036A). Using RNU6 B as control, 2μl each sample reverse transcription products were taken with application SYBR Premix Ex TaqTMⅡ(TaKaRa, Code No.RR820A). The products were formulated into liquid 25μl PCR reaction system using ABI 7500 instrument by TaqMan real-time quantitative polymerase chain reaction probe(qRT-PCR) and dual-specific PCR primers to detect the Ct value of mi R-133 a, mi R-208 a, mi R-499 a with RNU6 B as internal control following the instructions.5. Statistical analysis: △Ct value of each sample was calculated using SPSS19.0. Normally distributed data and clinical data of experimental data were presented as Mean ± Standard Deviation(x±s). Mann-Whitney U test was used to compare differences between two sets of skewed distribution data. The mi RNA levels of two groups were compared using independent sample t-test, and mi RNA levels of multiple groups were compared using chi-square test. Correlation between the two variables was tested using Pearson correlation analysis. P < 0.05 was considered significant statistical different. Results:1. Compared with the control group, there was a significant difference in the smoking history and high density lipoprotein(HDL) levels in the patients with CHD(P < 0.05). Compared with the control group, there was higher proportion of smoking patients and lower HDL levels in the CHD group. Coronary heart disease group and control group had no difference(P > 0.05) in age or sex, body weight, body mass index(BMI), triglyceride(TG), total cholesterol(TC), fasting blood glucose, hypertension, low density lipoprotein(LDL), history of diabetes, serum creatinine, uric acid.2. According to the Gensini score, all cases were divided into four groups, and each group had statistically significant difference(P < 0.05) in the level of triglyceride(TG), total cholesterol(TC), and low density lipoprotein(LDL). and the four groups show ed no difference(P > 0.05) in age, sex, weight, body mass index(BMI), history of hypertension, diabetes, smoking history, fasting blood sugar, high density lipoprotein(LDL), creatinine, uric acid.3. Compared with the control group, Real time fluorescent quantitative PCR show that the levels of mi RNA-133 a, mi RNA-208 a and mi RNA-499 a in CHD group had significant increased(P < 0.05).4. According to the Gensini score, all cases were divided into four groups, and the results detected by Real time fluorescent quantitative PCR show ed that the levels of mi RNA-133 a, mi RNA-208 a and mi RNA-499 a in each groups had statistically significant difference(P < 0.05) and the expression levels had positive correlation with Gensini score which presented the degree of coronary artery stenosis. Conclusions:The levels of plasma mi RNA-133 a,mi RNA-208 a and mi RNA-499 a in CHD groups were significantly higher than those in control group. With the increase of degree of coronary artery stenosis, the levels of plasma mi RNA-133 a, mi RNA-208 a and mi RNA-499 a were statistically significant difference among groups. The expression levels of these miRNAs had positive correlation with Gensini score which presented the degree of coronary artery stenosis. |
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