MicroRNA-10a-5p在大鼠心衰发展中的作用和机制研究
发布时间：2018-09-13 15:48
【摘要】：目的探讨micro RNA-10a-5p在大鼠心梗后心衰发生发展过程中的变化及其作用机制,并明确其在大鼠心肌细胞凋亡中发挥的作用。方法对健康SD大鼠行冠状动脉左前降支结扎(LAD)术建立心梗后心衰模型。通过监测大鼠体重并用超声心动图测量并计算大鼠左室短轴缩短率和左室射血分数等指标确定心衰模型的建立成功。培养出生1-3天的SD乳鼠心室肌细胞,苯肾上腺素(PE)诱导心肌细胞肥大建立心肌细胞肥大模型,通过观察细胞表面积,测量心肌肥大标志性基因表达变化等指标确定肥大模型的成功建立。脂质体转染人工合成的大鼠miRNA-10a-5p模拟物或抑制剂,使心肌细胞过表达miR-10a-5p或抑制其miR-10a的表达。实时荧光定量PCR(q RT-PCR)检测miR-10a-5p的表达量及ANP,BNP等心衰标志分子的m RNA水平表达量。Western Blot方法检测心衰相关蛋白,凋亡相关蛋白及靶蛋白的表达量。生物信息学预测miR-10a-5p的候选靶基因,采用双荧光报告基因检测系统检测荧光素酶的表达量,反映miRNA在离体体系中能否调控此基因表达。结果一、miR-10a-5p的表达量在心衰模型中升高采用LAD成功建立大鼠心衰模型,心衰组(Mol)大鼠相比于假手术组(Sham)大鼠,体重下降,活动减少,精神状态差,毛发无光泽。结扎后,LVFS降低了近50%(P0.01),LVEF降低了约35%(P0.01)。相比于Sham组,Mol-I组(Mol组心肌梗死区)的ANP表达量增高约75倍(P0.01),而BNP含量增高约17倍(P0.01)。相比于Sham组,Mol-R(Mol组心肌非梗死区)组的ANP表达了增高约20倍(P0.01),而BNP含量增高约15倍(P0.05)。结扎组大鼠心脏梗死区(Mol-I)ANP m RNA水平表达量比Sham组增高了约75倍(P0.01),结扎组大鼠非梗死区(Mol-R)ANP m RNA水平表达量比Sham组增高约20倍(P0.01);如图1-3所示,Mol-I组BNP的m RNA水平表达量比Sham组增高约17倍(P0.01),Mol-R组BNP m RNA水平表达量比Sham组增高约15倍(P0.05)。Mol组miR-10a-5p表达量比于Sham组增高了1.8倍(P0.05)。二、明确GATA6是miR-10a-5p的靶基因生物信息学方法筛选出GATA6是miR-10a-5p的候选靶基因,双荧光素酶报告基因结果显示,miR-10a-5p能直接抑制GATA6的表达。Western Blot结果显示,无论在心衰大鼠模型中还是在心肌肥大细胞中,miR-10a-5p和GATA6蛋白水平的变化都是相反的。过表达miR-10a-5p后,GATA6蛋白显著降低。三、miR-10a-5p的表达量在心肌细胞肥大模型中降低PE诱导心肌细胞成功的建立了心肌细胞肥大模型。相比于CON组,PE50组ANP,BNP的表达量均增高,但无统计学差异。相对于CON组,PE100组的ANP表达量增加了约18倍(P0.01),BNP的表达量增加了约88倍(P0.05);相对于CON组,PE200组的ANP表达量增加了约37.5倍(P0.01),而BNP增加了160多倍(P0.01)。PE组较CON组细胞表面积显著增加。PE组较CON组,miR-10a-5p表达量降低(P0.01)四、miR-10a-5p促进大鼠心肌细胞凋亡PI单染后,荧光显微镜观察结果显示,过表达miR-10a-5p后,凋亡细胞个数显著增加;抑制miR-10a-5p的表达,凋亡细胞个数降低。Western Blot结果显示,过表达miR-10a-5p能增加cleaved-caspase3的蛋白表达量,抑制Bcl-2的蛋白表达量。结论本研究通过大鼠左冠状动脉前降支结扎所建立的大鼠心梗后心衰模型和苯肾上腺素刺激所建立的体外培养的大鼠乳鼠心肌细胞肥大模型,利用分子生物学,细胞生物学和生物信息学的方法,明确了1:心衰大鼠中miR-10a-5p的表达量升高,这种高表达的miR-10a-5p可以通过负性调控下游靶基因GATA6促进心衰;2:过表达miR-10a-5p能够促进心肌细胞凋亡。
[Abstract]:Objective To investigate the changes and mechanism of micro RNA-10a-5p in the development of heart failure after myocardial infarction in rats and its role in myocardial apoptosis. The cardiac hypertrophy model of SD neonatal rats was established by culturing ventricular myocytes from 1 to 3 days old and inducing cardiomyocyte hypertrophy by phenylephrine (PE). By observing the cell surface area and measuring the changes of gene expression markers of cardiac hypertrophy, the cardiac hypertrophy model was established. Liposome transfection of synthetic rat microRNAs-10a-5p mimics or inhibitors induced overexpression of microRNAs-10a-5p in cardiomyocytes or inhibited the expression of microRNAs-10a-5p in cardiomyocytes. Real-time fluorescence quantitative PCR (q RT-PCR) was used to detect the expression of microRNAs-10a-5p and the expression of microRNAs of heart failure markers such as ANP and BNP. Methods The expression of HF-related proteins, apoptosis-related proteins and target proteins were detected. Bioinformatics predicted the candidate target gene of microRNA10a-5p, and the expression of luciferase was detected by double fluorescent reporter gene detection system. Results 1. The expression of microRNAs in HF model was detected by microRNAs. After ligation, LVFS decreased by nearly 50% (P 0.01), LVEF decreased by about 35% (P 0.01). Compared with Sham group, the expression of ANP increased by about 75% in Mol-I group (myocardial infarction area). Compared with the Sham group, the expression of ANP in the non-infarct area of the Mol-R group was about 20 times higher (P 0.01), while the content of BNP was about 15 times higher (P 0.05). The expression of ANP-m RNA in the myocardial infarct area (Mol-I) of the ligated group was about 75 times higher than that in the Sham group (P 0.01). The expression level of PMRNA in Mol-I group was about 17 times higher than that in Sham group (P 0.01), and that in Mol-R group was about 15 times higher than that in Sham group (P 0.05). The expression level of Mi-10a-5p in Mol group was 1.8 times higher than that in Sham group (P 0.05). GATA6 was screened as a candidate target gene for microarray-10a-5p by bioinformatics. The results of double luciferase reporter gene showed that microarray-10a-5p could directly inhibit the expression of GATA6. Western Blot results showed that the changes of the levels of microarray-10a-5p and GATA6 protein were opposite in heart failure rat models and in cardiac mast cells. Compared with CON group, the expression of ANP and BNP in PE50 group increased, but there was no significant difference. Compared with CON group, the expression of ANP in PE100 group increased about 18 times (P 0.01). Compared with CON group, the expression of ANP in PE200 group increased about 37.5 times (P 0.01), while that in BNP group increased more than 160 times (P 0.01). Compared with CON group, the cell surface area of PE group increased significantly. Compared with CON group, the expression of microwave-10a-5p decreased (P 0.01) 4 in PE group, and the expression of microwave-10a-5p promoted apoptosis of rat cardiomyocytes after PI single staining, the fluorescence was obvious. Western Blot results showed that overexpression of microRNAs-10a-5p could increase the expression of cleaved-caspase 3 protein and inhibit the expression of Bcl-2 protein in the left anterior descending coronary artery of rats. The rat model of heart failure after myocardial infarction induced by ligation and the rat model of hypertrophy induced by phenylephrine in vitro were established. Molecular biology, cell biology and bioinformatics methods were used to determine the increased expression of microRNA-10a-5p in 1:HF rats. This high expression of microRNA-10a-5p could be negatively expressed. Regulation of downstream target gene GATA6 promotes heart failure; 2: over expression of miR-10a-5p can promote cardiomyocyte apoptosis.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.6
本文编号：2241621
[Abstract]:Objective To investigate the changes and mechanism of micro RNA-10a-5p in the development of heart failure after myocardial infarction in rats and its role in myocardial apoptosis. The cardiac hypertrophy model of SD neonatal rats was established by culturing ventricular myocytes from 1 to 3 days old and inducing cardiomyocyte hypertrophy by phenylephrine (PE). By observing the cell surface area and measuring the changes of gene expression markers of cardiac hypertrophy, the cardiac hypertrophy model was established. Liposome transfection of synthetic rat microRNAs-10a-5p mimics or inhibitors induced overexpression of microRNAs-10a-5p in cardiomyocytes or inhibited the expression of microRNAs-10a-5p in cardiomyocytes. Real-time fluorescence quantitative PCR (q RT-PCR) was used to detect the expression of microRNAs-10a-5p and the expression of microRNAs of heart failure markers such as ANP and BNP. Methods The expression of HF-related proteins, apoptosis-related proteins and target proteins were detected. Bioinformatics predicted the candidate target gene of microRNA10a-5p, and the expression of luciferase was detected by double fluorescent reporter gene detection system. Results 1. The expression of microRNAs in HF model was detected by microRNAs. After ligation, LVFS decreased by nearly 50% (P 0.01), LVEF decreased by about 35% (P 0.01). Compared with Sham group, the expression of ANP increased by about 75% in Mol-I group (myocardial infarction area). Compared with the Sham group, the expression of ANP in the non-infarct area of the Mol-R group was about 20 times higher (P 0.01), while the content of BNP was about 15 times higher (P 0.05). The expression of ANP-m RNA in the myocardial infarct area (Mol-I) of the ligated group was about 75 times higher than that in the Sham group (P 0.01). The expression level of PMRNA in Mol-I group was about 17 times higher than that in Sham group (P 0.01), and that in Mol-R group was about 15 times higher than that in Sham group (P 0.05). The expression level of Mi-10a-5p in Mol group was 1.8 times higher than that in Sham group (P 0.05). GATA6 was screened as a candidate target gene for microarray-10a-5p by bioinformatics. The results of double luciferase reporter gene showed that microarray-10a-5p could directly inhibit the expression of GATA6. Western Blot results showed that the changes of the levels of microarray-10a-5p and GATA6 protein were opposite in heart failure rat models and in cardiac mast cells. Compared with CON group, the expression of ANP and BNP in PE50 group increased, but there was no significant difference. Compared with CON group, the expression of ANP in PE100 group increased about 18 times (P 0.01). Compared with CON group, the expression of ANP in PE200 group increased about 37.5 times (P 0.01), while that in BNP group increased more than 160 times (P 0.01). Compared with CON group, the cell surface area of PE group increased significantly. Compared with CON group, the expression of microwave-10a-5p decreased (P 0.01) 4 in PE group, and the expression of microwave-10a-5p promoted apoptosis of rat cardiomyocytes after PI single staining, the fluorescence was obvious. Western Blot results showed that overexpression of microRNAs-10a-5p could increase the expression of cleaved-caspase 3 protein and inhibit the expression of Bcl-2 protein in the left anterior descending coronary artery of rats. The rat model of heart failure after myocardial infarction induced by ligation and the rat model of hypertrophy induced by phenylephrine in vitro were established. Molecular biology, cell biology and bioinformatics methods were used to determine the increased expression of microRNA-10a-5p in 1:HF rats. This high expression of microRNA-10a-5p could be negatively expressed. Regulation of downstream target gene GATA6 promotes heart failure; 2: over expression of miR-10a-5p can promote cardiomyocyte apoptosis.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.6
【参考文献】
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1 季红斌,翟琦巍,刘新垣,郑仲承;bcl-2基因的转录调控[J];生物化学与生物物理学报;2000年02期
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