血液循环中与急性心肌梗死相关联miRNA的探索

发布时间：2018-05-03 20:00

本文选题：急性心肌梗死 + miR-486　；参考：《郑州大学》2016年博士论文

【摘要】：背景和目的心血管疾病因其逐年增长的发病率和死亡率,一直以来受到各界广泛关注。尽管目前已有广泛可用的诊断治疗途径,心血管疾病的流行、死亡率和治疗花费在发达国家及发展中国家仍呈上升趋势。急性心肌梗死(acute myocardial infarction,AMI)是世界范围内最常见的心血管疾病之一,为有效控制AMI的发生和发展,寻求新的诊断和治疗的指标和方法十分必要。近年以来,研究表明mi RNA在疾病发生发展中可通过靶向调节其下游靶基因发挥作用。在多个研究领域都得到广泛重视,包括肿瘤、心血管疾病等等。已发现多种mi RNA在急性心肌梗死患者血液和血浆中发生改变,如mi R-1、mi R-133a,mi R-208b,mi R-499,和mi R-328,提示循环mi RNA在AMI早期诊断中具有价值。本实验的目的是筛选AMI发生时血液循环中异常表达的mi RNA;分析异常表达mi RNA表达水平变化,并判断其作为辅助诊断的价值;初步探索AMI发生时异常表达mi RNA发挥调控作用的可能途径。第一部分急性心肌梗死发生时血液循环中异常表达mi RNA筛选和鉴定方法1.收集30例AMI患者和30例健康成人志愿者的血浆样本。2.采用Agilent mi RNA芯片分别检测随机选取3例AMI患者和3例健康成年志愿者血浆中mi RNA的表达情况,筛选有显著差异表达的mi RNA。3.应用q RT-PCR的方法检测30例AMI患者和30例健康成人志愿者的血浆样本中mi RNA芯片筛选出的有差异表达的mi RNA的表达情况,对mi RNA芯片的结果进行验证。4.应用统计学软件SPSS21.0对实验数据进行统计学分析,检验标准α=0.05。结果1.通过mi RNA芯片检测出共有36种mi RNA在AMI患者血浆中出现异常表达,其中有23种mi RNA表达水平显著升高(P0.05),13种mi RNA表达水平明显下降(P0.05)。2.q RT-PCR验证的结果表明30例AMI患者血浆中除mi R-361-5p,mi R-182-5p,mi R-497-5p,mi R-20a-5p四种mi RNA表达水平与正常对照组相比无显著性差异(P0.05),其余32种mi RNA表达水平较正常对照组相比出现显著性变化(P0.05),其中上调的mi RNA有20种,下调的mi RNA有12种。第二部分AMI发生时血液循环中mi R-486,mi R-150和mi R-26a表达变化分析及作为辅助诊断的价值方法1.收集110例AMI和110例健康成人志愿者的血浆样本,110例AMI患者中分别有45例NSTEMI患者和65例STEMI患者,采集入院之后发病4h以内,24h,48h,72h的血浆样本。2.q RT-PCR法检测mi R-486,mi R-150和mi R-26a在AMI各类型各时间段血浆样本中的表达水平。3.应用受试者工作特性曲线(ROC)反映灵敏度与特异度,应用ROC曲线下面积(AUCROC)比较mi R-486,mi R-150和mi R-26a对AMI的辅助诊断价值。4.应用统计学软件SPSS21.0处理所有数据,检验标准α=0.05。结果1.AMI发作4h以内,mi R-486和mi R-150表达量分别为9.655±1.427和7.420±0.911,与正常对照组相比表达水平均显著上调(P0.05);mi R-26a表达量为0.092±0.104,与正常对照组相比表达水平显著下调(P0.05)。2.AMI发作4h以内,24h,48h,72h,mi R-486和mi R-150表达水平均随时间延长逐渐降低,至72h时与正常对照组无显著性差异(P0.05);mi R-26a表达水平随时间延长逐渐升高,至72h时与正常对照组无显著性差异(P0.05)。3.AMI发作4h以内,mi R-486,mi R-150和mi R-26a的AUC曲线下面积分别为0.731(P0.05),0.678(P0.05),0.763(P0.05),表明这三种mi RNA的表达水平检测对检测AMI的发生有一定的价值。这三种mi RNA联合检测的AUC曲线下面积为0.792(P0.05),表明具有更高的诊断价值。4.AMI发作4h以内,mi R-486,mi R-150和mi R-26a表达在AMI的两种类型STEMI和NSTEMI中存在显著性差异。mi R-486,mi R-150和mi R-26a在STEMI中的AUC曲线下面积分别为0.695(P0.05),0.639(P0.05),0.750(P0.05),表明这三种mi RNA的表达水平检测对检测STEMI的发生有一定的价值;mi R-486,mi R-150和mi R-26a在NSTEMI中的AUC曲线下面积分别为0.782(P0.05),0.734(P0.05),0.782(P0.05),表明这三种mi RNA的表达水平检测对检测NSTEMI的发生有一定的价值;在STEMI和NSTEMI时这三种mi RNA联合检测的AUC曲线下面积分别为0.765(P0.05),0.833(P0.05),说明三者联合检测具有更高的灵敏度和特异度,且在NSTEMI时具有更高的诊断价值。第三部分mi R-486,mi R-150在AMI发生时的作用机制的初步探索方法1.通过Target Scan和mi Randa寻找可能与mi R-486,mi R-150相关的靶基因2.用Overlap PCR的方法扩增突变型CDKN1B 3’UTR和ALDH2 3’UTR片段,用普通PCR来扩增野生型CDKN1B 3’UTR和ALDH2 3’UTR片段,将扩增出来的片段与pmir GLO双粘载体进行重组,从而分别构建野生型和突变型CDKN1B 3’UTR和ALDH2 3’UTR的重组双荧光素酶报告载体。3.将野生型和突变型CDKN1B 3’UTR重组报告载体分别与mi R-150 mimics或者mi R-150 scramble共同转染到HEK 293T细胞中;将野生型和突变型ALDH2 3’UTR重组报告载体分别与mi R-486 mimics或者mi R-486 scramble共同转染到HEK 293T细胞中。通过双荧光素酶报告实验验证CDKN1B是否为mi R-150的靶基因,ALDH2是否为mi R-486的靶基因。4.应用统计学软件SPSS21.0处理所有数据,检验标准α=0.05。结果1.生物信息学检测发现基因CDKN1B的3’UTR区可能是mi R-150的一个下游直接作用靶点,ALDH2的3’UTR区可能是mi R-486的一个下游直接作用靶点。2.经酶切验证,PCR验证和测序结果验证,成功构建了野生型和突变型CDKN1B 3’UTR和ALDH2 3’UTR的重组双荧光素酶报告载体。3.双荧光素报告酶实验显示,mi R-150 mimics和WT-pmir GLO-CDKN1B共转染组荧光素酶活性为0.54±0.065,显著低于其他三个共转染组(P0.05),表明CDKN1B是mi R-150作用的靶基因;mi R-486 mimics和WT-pmir GLOALDH2共转染组荧光素酶活性为0.62±0.045,显著低于其他三个共转染组(P0.05),表明ALDH2是mi R-486作用的靶基因。结论1.急性心肌梗死患者循环血液中,存在32种mi RNA出现表达异常,20种mi RNA表达明显上调,12种mi RNA表达明显下调。2.急性心肌梗死发作4h以内,mi R-486和mi R-150表达水平明显上调,mi R-26a表达水平明显下调,且随时间延长变化趋势越不明显。mi R-486,mi R-150和mi R-26a联合检测有望成为急性心肌梗死的辅助诊断指标,尤其适用于NSTEMI的辅助诊断。3.mi R-150和mi R-486分别负向调节CDKN1B和ALDH2的表达,可能在急性心肌梗死发生发展中发挥一定作用。
[Abstract]:Background and objective cardiovascular disease has been widely concerned because of its increasing incidence and mortality year by year. Despite the widespread available diagnostic methods, the prevalence of cardiovascular disease, mortality and treatment costs are still rising in developed and developing countries. Acute myocardial infarction (acute myocardial) Infarction, AMI) is one of the most common cardiovascular diseases in the world. In order to effectively control the occurrence and development of AMI, it is necessary to seek new diagnostic and therapeutic targets and methods. In recent years, studies have shown that MI RNA can play a role in regulating the target gene by targeting the target gene in the development of the disease. In many fields, it has been obtained. A variety of MI RNA have been found in the blood and plasma of patients with acute myocardial infarction, such as mi R-1, MI R-133a, MI R-208b, MI R-499, and MI R-328. Mi RNA; analyze abnormal expression of MI RNA expression level, and judge its value as auxiliary diagnosis; preliminary explore the possible way of abnormal expression of MI RNA when AMI occurs. In part 1, abnormal expression of MI RNA screening and identification in blood circulation during the occurrence of acute myocardial infarction 1., 30 cases of AMI patients and 30 healthy adult cases were collected. The plasma sample.2. of human volunteers was selected by Agilent mi RNA chip to detect the expression of MI RNA in plasma of 3 AMI patients and 3 healthy adult volunteers, and to screen the MI RNA.3. application Q RT-PCR method with significant differential expression for the detection of 30 cases of AMI patients and 30 healthy adult volunteers. The expression of the differentially expressed mi RNA, the results of the MI RNA chip were verified by the.4. application statistics software SPSS21.0 for statistical analysis of the experimental data. The test standard alpha =0.05. result 1. detected a total of 36 mi RNA in the AMI patients' plasma by Mi RNA chip, and there were 23 kinds of expression levels. (P0.05), the expression level of 13 kinds of MI RNA decreased significantly (P0.05).2.q RT-PCR verification. The results showed that there were no significant differences in the levels of MI R-361-5p, MI R-182-5p, MI, and four kinds of expressions in the plasma of the AMI patients compared with the normal control group, and the other 32 kinds of expressions were compared with the normal control group. Significant changes (P0.05), among which there were 20 kinds of MI RNA, 12 down regulated mi RNA, MI R-486, MI R-150 and MI R-26a in the blood circulation during the occurrence of AMI, and the value method of auxiliary diagnosis. 1. the plasma samples of 110 cases and 110 healthy adult volunteers were collected, and 45 of 110 patients were respectively. Patients and 65 patients with STEMI were collected after admission to 4h, 24h, 48h, and 72h plasma samples were detected by.2.q RT-PCR method for the detection of MI R-486, MI R-150 and Mi expressions in each type of plasma samples. The auxiliary diagnostic value of R-486, MI R-150 and MI R-26a for AMI.4. applied statistics software SPSS21.0 to process all the data, and to test the standard alpha =0.05. results within 1.AMI 4h, 9.655 + 1.427 and 7.420 + 0.911 respectively, compared with the normal control group. The expression level of 24h, 48h, 72h, MI R-486 and MI R-150 decreased gradually with time, and there was no significant difference between the normal control group and the normal control group. There was no significant difference between the normal control group and the normal control group. There was no significant difference between the normal control group and the normal control group. The expression level of 24h, 48h, 72h, MI R-486 and MI R-150 increased gradually. The area under the AUC curve of MI R-486, MI R-150 and MI R-26a is 0.731 (P0.05), 0.678 (P0.05) and 0.763 (0.763), indicating that the three kinds of expression levels have a certain value for the occurrence of the detection. The area under the three combined detection curves is 0.792, indicating a higher level of 4H. The diagnostic value of.4.AMI is within 4h, MI R-486, MI R-150 and MI R-26a have significant differences in the two types of STEMI and NSTEMI AMI. The area under the curve is 0.695, 0.639, 0.750. The values of MI R-486, MI R-150 and MI R-26a under AUC curves in NSTEMI are 0.782 (P0.05), 0.734 (P0.05), and 0.782 (P0.05). P0.05), 0.833 (P0.05), indicating that the combined detection of the three has a higher sensitivity and specificity, and has a higher diagnostic value at NSTEMI. Third part of the MI R-486, MI R-150 is a preliminary exploration of the mechanism of the occurrence of AMI. 1. through Target Scan and MI Randa. The mutant CDKN1B 3 'UTR and ALDH2 3' UTR fragment were amplified by R, and the wild type CDKN1B 3 'UTR and ALDH2 3' UTR fragments were amplified by ordinary PCR, and the amplified fragments were reorganized with the pmir GLO double adhered carrier to construct the recombinant double luciferase reporter vector of wild type and mutant 3 '3' and 3 'respectively. The wild type and mutant CDKN1B 3 'UTR recombinant report vectors were transfected to HEK 293T cells, respectively, with MI R-150 mimics or MI R-150 scramble, respectively. The experiment verifies whether CDKN1B is the target gene of MI R-150, and whether ALDH2 is the target gene of MI R-486.4. applied statistics software SPSS21.0 to deal with all data and test the standard alpha =0.05. result 1. bioinformatics detection found that the 3 'UTR region of the gene CDKN1B may be a downstream direct target of MI. A direct targeting target of downstream.2. was verified by enzyme digestion and verified by PCR and sequencing results. The recombinant double luciferase reporter of wild type and mutant CDKN1B 3 'UTR and ALDH2 3' UTR was successfully constructed,.3. double luciferase reporter experiment showed that the luciferase activity of MI R-150 mimics and WT-pmir GLO-CDKN1B co transfected group was 0.54 + 0.. 065, significantly lower than the other three co transfection groups (P0.05), indicating that CDKN1B was the target gene for MI R-150, and the luciferase activity of MI R-486 mimics and WT-pmir GLOALDH2 co transfection group was 0.62 + 0.045, significantly lower than the other three co transfected groups (P0.05), indicating that ALDH2 was the target gene of MI. The expression of 32 kinds of MI RNA was abnormal, the expression of the 20 mi RNA was obviously up-regulated, the expression of the 12 mi RNA was obviously down regulated within 4H of the acute myocardial infarction, and the expression level of MI R-486 and MI R-150 was obviously down regulated. It is expected to be an auxiliary diagnostic indicator for acute myocardial infarction, especially for the auxiliary diagnosis of NSTEMI,.3.mi R-150 and MI R-486, which can negatively regulate the expression of CDKN1B and ALDH2, and may play a role in the development of acute myocardial infarction.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.22

【参考文献】