MicroRNA-10a-5p在大鼠心衰發(fā)展中的作用和機制研究
發(fā)布時間：2018-09-13 15:48
【摘要】：目的探討micro RNA-10a-5p在大鼠心梗后心衰發(fā)生發(fā)展過程中的變化及其作用機制,并明確其在大鼠心肌細胞凋亡中發(fā)揮的作用。方法對健康SD大鼠行冠狀動脈左前降支結(jié)扎(LAD)術(shù)建立心梗后心衰模型。通過監(jiān)測大鼠體重并用超聲心動圖測量并計算大鼠左室短軸縮短率和左室射血分數(shù)等指標(biāo)確定心衰模型的建立成功。培養(yǎng)出生1-3天的SD乳鼠心室肌細胞,苯腎上腺素(PE)誘導(dǎo)心肌細胞肥大建立心肌細胞肥大模型,通過觀察細胞表面積,測量心肌肥大標(biāo)志性基因表達變化等指標(biāo)確定肥大模型的成功建立。脂質(zhì)體轉(zhuǎn)染人工合成的大鼠miRNA-10a-5p模擬物或抑制劑,使心肌細胞過表達miR-10a-5p或抑制其miR-10a的表達。實時熒光定量PCR(q RT-PCR)檢測miR-10a-5p的表達量及ANP,BNP等心衰標(biāo)志分子的m RNA水平表達量。Western Blot方法檢測心衰相關(guān)蛋白,凋亡相關(guān)蛋白及靶蛋白的表達量。生物信息學(xué)預(yù)測miR-10a-5p的候選靶基因,采用雙熒光報告基因檢測系統(tǒng)檢測熒光素酶的表達量,反映miRNA在離體體系中能否調(diào)控此基因表達。結(jié)果一、miR-10a-5p的表達量在心衰模型中升高采用LAD成功建立大鼠心衰模型,心衰組(Mol)大鼠相比于假手術(shù)組(Sham)大鼠,體重下降,活動減少,精神狀態(tài)差,毛發(fā)無光澤。結(jié)扎后,LVFS降低了近50%(P0.01),LVEF降低了約35%(P0.01)。相比于Sham組,Mol-I組(Mol組心肌梗死區(qū))的ANP表達量增高約75倍(P0.01),而BNP含量增高約17倍(P0.01)。相比于Sham組,Mol-R(Mol組心肌非梗死區(qū))組的ANP表達了增高約20倍(P0.01),而BNP含量增高約15倍(P0.05)。結(jié)扎組大鼠心臟梗死區(qū)(Mol-I)ANP m RNA水平表達量比Sham組增高了約75倍(P0.01),結(jié)扎組大鼠非梗死區(qū)(Mol-R)ANP m RNA水平表達量比Sham組增高約20倍(P0.01);如圖1-3所示,Mol-I組BNP的m RNA水平表達量比Sham組增高約17倍(P0.01),Mol-R組BNP m RNA水平表達量比Sham組增高約15倍(P0.05)。Mol組miR-10a-5p表達量比于Sham組增高了1.8倍(P0.05)。二、明確GATA6是miR-10a-5p的靶基因生物信息學(xué)方法篩選出GATA6是miR-10a-5p的候選靶基因,雙熒光素酶報告基因結(jié)果顯示,miR-10a-5p能直接抑制GATA6的表達。Western Blot結(jié)果顯示,無論在心衰大鼠模型中還是在心肌肥大細胞中,miR-10a-5p和GATA6蛋白水平的變化都是相反的。過表達miR-10a-5p后,GATA6蛋白顯著降低。三、miR-10a-5p的表達量在心肌細胞肥大模型中降低PE誘導(dǎo)心肌細胞成功的建立了心肌細胞肥大模型。相比于CON組,PE50組ANP,BNP的表達量均增高,但無統(tǒng)計學(xué)差異。相對于CON組,PE100組的ANP表達量增加了約18倍(P0.01),BNP的表達量增加了約88倍(P0.05);相對于CON組,PE200組的ANP表達量增加了約37.5倍(P0.01),而BNP增加了160多倍(P0.01)。PE組較CON組細胞表面積顯著增加。PE組較CON組,miR-10a-5p表達量降低(P0.01)四、miR-10a-5p促進大鼠心肌細胞凋亡PI單染后,熒光顯微鏡觀察結(jié)果顯示,過表達miR-10a-5p后,凋亡細胞個數(shù)顯著增加;抑制miR-10a-5p的表達,凋亡細胞個數(shù)降低。Western Blot結(jié)果顯示,過表達miR-10a-5p能增加cleaved-caspase3的蛋白表達量,抑制Bcl-2的蛋白表達量。結(jié)論本研究通過大鼠左冠狀動脈前降支結(jié)扎所建立的大鼠心梗后心衰模型和苯腎上腺素刺激所建立的體外培養(yǎng)的大鼠乳鼠心肌細胞肥大模型,利用分子生物學(xué),細胞生物學(xué)和生物信息學(xué)的方法,明確了1:心衰大鼠中miR-10a-5p的表達量升高,這種高表達的miR-10a-5p可以通過負性調(diào)控下游靶基因GATA6促進心衰;2:過表達miR-10a-5p能夠促進心肌細胞凋亡。
[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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.6
本文編號：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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.6
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相關(guān)期刊論文 前1條
1 季紅斌,翟琦巍,劉新垣,鄭仲承;bcl-2基因的轉(zhuǎn)錄調(diào)控[J];生物化學(xué)與生物物理學(xué)報;2000年02期
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