MiR-34a在高血压诱导血管损伤中的表达及其作用机制研究
发布时间：2018-08-23 14:13
【摘要】：研究背景原发性高血压(primary hypertension)是临床常见的一种慢性疾病,也是心脑血管病最主要的危险因素,其主要并发症如脑卒中、心肌梗死、心力衰竭及慢性肾病等,不仅致残、致死率高,而且严重消耗医疗和社会资源,给家庭和社会造成沉重负担,对人类健康危害较大,其发病机制目前仍不明确。近年来,随着我国人口老龄化、生活水平的改善、生活节奏加快等因素,原发性高血压发病率持续升高,且年轻患者比例显著增多,原发性高血压的预防和治疗引起了研究者们高度重视。血管损伤是高血压疾病的基本病理改变之一,主要包括血管内皮细胞损伤、平滑肌细胞增殖和血管重构等病理过程。研究发现,血管内皮细胞损伤在高血压的发生发展中具有重要的作用。例如：高血压诱导的血管内皮细胞功能失调,破坏其NO与内皮素的分泌稳态,诱导血管收缩加强,从而增加了循环阻力,促进高血压的发展；在高血压环境中,微血管内皮细胞的凋亡率显著升高,导致血液平行通路减少,提高了外周循环阻力,导致血压升高。尽管越来越多的研究显示,高血压诱导的血管内皮细胞损伤在高血压发生发展中具有重要作用,但其分子机理目前尚不明确,仍有待进一步研究。MicroRNA(miRNA)是一类长度为18-24核苷酸左右的非编码小分子RNA,其进化呈高度保守,主要通过与靶基因mRNA的3口非翻译区(3□ untranslated region,3□-UTR)区完全或不完全结合,在转录后水平通过抑制蛋白质翻译或mRNA降解调控基因的表达。目前研究表明,高血压患者外周血中miRNA表达谱发生了明显改变,甚至具有高血压早期诊断和预后判断的临床价值。此外,在高血压引起的并发症中, miRNA也发挥着重要的生物学功能。此外,多种miRNA分子也参与了血管内皮细胞损伤。MiR-34a是一个多功能调控子,通过对相关靶基因的表达调控,参与细胞分裂、衰老、凋亡和增殖的调控。然而,1miRNA-34a在高血压中的表达及作用机制仍不明确。在本研究中,我们首先检测miR-34a在高血压病患者外周血表达情况及其与高血压病临床病理特征之间的关系；然后,从体外实验方面进行深入研究,探讨miR-34a在高血压内皮细胞损伤中的作用及其机制,这可能为高血压的临床治疗提供新的治疗靶点。目的研究miR-34a在高血压患者外周血中的表达及在高血压诱导的血管内皮细胞损伤中的分子调控机制。方法1.收集50例原发性高血压患者外周血及28例正常健康人外周血,采用实时定量PCR检测miR-34a的表达；2.将miR-34a抑制物(miR-34a inhibitor)以及其阴性对照(scramble control miRNA)分别转入人脐静脉内皮细胞(HUVEC)中,转染48h后进行下一步检测。3.分别采用CCK-8、Transwell及流式细胞术研究miR-34a分子对HUVECs的增殖、细胞周期、凋亡和迁移能力的影响。4.采用双荧光素酶报告实验鉴定Tgif2是否为miR-34a靶基因。5.采用Western blot检测下调miR-34a表达后,HUVECs中miR-34a潜在靶基因Tgif2的表达。6.采用SPSS16.0统计软件进行统计分析,所有计量数据均采用均数±标准差(x±s)表示,分组比较使用成组t检验,以P0.05有统计学意义。结果：1.MiR-34a在高血压患者外周血中的表达及临床意义实时荧光定量PCR结果显示,与正常对照组相比,高血压患者外周血中miR-34a的表达明显升高,差异有统计学意义(P0.05)；在临床病理特征分组比较中,高血压3期患者外周血miR-34a表达明显高于1期和2期患者(P0.05)；这一结果表明,高血压患者外周血中miR-34a的表达上调,且与高血压进展关系紧密。2. MiR-34a对HUVECs增殖能力的影响CCK-8实验结果显示,下调miR-34a表达后,血管内皮细胞]HUVECs的体外增殖能力明显增强,表明miR-34a具有抑制HUVECs的增殖的功能,其在高血压患者外周血中的高表达可能抑制血管内皮细胞的增殖。3. MiR-34a对HUVECs迁移能力的影响Transwell实验结果显示,下调miR-34a表达后, HUVECs细胞迁移能力明显升高,穿过小室膜的细胞数多于NC组(74.5±4.30 vs 48.5±6.3),差异有统计学意义(P0.05)；这一结果表明miR-34a能够抑制HUVECs细胞迁移能力,在高血压引起的血管内皮损伤中,可能导致邻近血管内皮细胞向损伤处的迁移受抑,从而影响损伤血管内皮细胞的修复。4. MiR-34a对HUVECs细胞周期的影响流式细胞术检测miR-34a对HUVECs细胞周期的影响,结果显示,与NC组相比,miR-34a inhibitor组的HUVECs出现了G1/S期转换加快,表明过表达miR-34a能够调控G1/S期转换从而抑制HUVECs的增殖,从而抑制血管内皮细胞的修复。5. MiR-34a对HUVECs凋亡的影响流式细胞仪检测结果显示,转染miR-34a inhibitor后,人脐静脉内皮细胞的凋亡率明显降低(P0.05),表明在高血压发生发展过程中,降低miR-34a的表达能够抑制HUVECs细胞凋亡,可能是高血压诱导的血管内皮损伤的作用机制之一。6. Tigf2为miR-34a的靶基因生物信息学分析显示,miR-34a和Tgif2的3口-UTR区存在结合位点,我们采用双荧光素酶报告基因实验来确定Tgif2是否为miR-34a的直接靶基因。结果表明,与NC组相比,共转染miR-34a mimics和Tigf2-wild type荧光素酶报告质粒后,荧光值显著下调(P0.05)；而Tigf2-mutant与miR-34a mimics共转后,荧光值并无明显差异(P0.05),表明miR-34a能与靶基因Tgif2 mRNA的3口-UTR种子区结合。免疫印迹结果显示,下调1miR-34a表达水平后,HUVECs中Tigf2蛋白表达均出现明显上升(P0.05)；提示miR-34a的生物学功能可能与Tigf2表达相关。结论：1. MiR-34a在高血压患者外周血中表达显著升高,且与高血压临床分级呈正相关；2. MiR-34a能够通过下调Tgif2表达抑制HUVECs细胞的增殖和迁移能力,促进其凋亡,从而促进血管内皮的损伤。
[Abstract]:Background Essential hypertension is a common chronic disease and the most important risk factor of cardiovascular and cerebrovascular diseases. Its main complications, such as stroke, myocardial infarction, heart failure and chronic kidney disease, not only cause disability and high mortality, but also seriously consume medical and social resources to create family and society. In recent years, with the aging of the population, the improvement of living standards, the quickening pace of life and other factors, the incidence of essential hypertension continues to rise, and the proportion of young patients significantly increased, the prevention and treatment of essential hypertension caused researchers. Vascular injury is one of the basic pathological changes in hypertension, including vascular endothelial cell injury, smooth muscle cell proliferation and vascular remodeling. It has been found that vascular endothelial cell injury plays an important role in the development of hypertension. It can disrupt the homeostasis of NO and ET secretion and induce vasoconstriction, which increases circulatory resistance and promotes the development of hypertension. In hypertensive environments, the apoptosis rate of microvascular endothelial cells increases significantly, leading to a decrease in blood parallel pathways, an increase in peripheral circulation resistance, and an increase in blood pressure. MicroRNA (microRNA) is a kind of non-coding small RNA with a length of 18-24 nucleotides, which is highly conserved and mainly through three ports of target gene mRNA. Complete or incomplete binding of the untranslated region (3_-UTR) region to regulate gene expression at the posttranscriptional level by inhibiting protein translation or mRNA degradation. Current studies have shown that the expression profile of microRNAs in the peripheral blood of hypertensive patients has changed significantly, and even has high clinical value in early diagnosis and prognosis of hypertension. MiR-34a is a multifunctional regulator involved in the regulation of cell division, senescence, apoptosis and proliferation by regulating the expression of related target genes. In this study, we first examined the expression of microRNAs in the peripheral blood of patients with hypertension and its relationship with the clinicopathological characteristics of hypertension; then, we conducted in-depth studies in vitro to explore the role and mechanism of microRNAs in the injury of hypertensive endothelial cells. Objective To investigate the expression of microRNA-34a in peripheral blood of patients with hypertension and its molecular regulation mechanism in vascular endothelial cell injury induced by hypertension. MicroRNA-34a inhibitor and scramble-controlled microRNA were transfected into human umbilical vein endothelial cells (HUVEC) for further detection 48 hours. CCK-8, Transwell and flow cytometry were used to study the proliferation, cell cycle, apoptosis of HUVECs. The expression of Tgif2 in HUVECs was detected by Western blot. 6. The expression of Tgif2 in HUVECs was analyzed by SPSS16.0 statistical software. All measurements were expressed by mean (+) standard deviation (x (+) s). Results: 1. The expression of MiR-34a in the peripheral blood of patients with hypertension and its clinical significance Real-time fluorescence quantitative PCR results showed that compared with the normal control group, the expression of MiR-34a in the peripheral blood of patients with hypertension was significantly higher, the difference was statistically significant (P 0.05). The expression of microRNA-34a in peripheral blood of patients with stage 3 hypertension was significantly higher than that of patients with stage 1 and 2 hypertension (P MiR-34a may inhibit the proliferation of vascular endothelial cells in hypertensive patients. 3. MiR-34a may affect the migration of HUVECs. Transwell experiment showed that HUVECs migration ability was inhibited by down-regulating the expression of MiR-34a. The number of cells passing through the ventricular membrane was significantly higher than that of NC group (74.5+4.30 vs 48.5+6.3), and the difference was statistically significant (P The effect of MiR-34a on the cell cycle of HUVECs was detected by flow cytometry. The results showed that G1/S phase transition of HUVECs in the inhibitor group was faster than that in the NC group, indicating that overexpression of MiR-34a could regulate G1/S phase transition and inhibit the proliferation of HUVECs, thereby inhibiting the proliferation of HUVECs. Effect of MiR-34a on apoptosis of HUVECs Flow cytometry showed that the apoptosis rate of HUVECs decreased significantly after transfection of MiR-34a inhibitor (P 0.05), suggesting that reducing the expression of MiR-34a could inhibit the apoptosis of HUVECs during the development of hypertension, which may be induced by hypertension. Tigf2 is a target gene for microRNAs-34a. Bioinformatics analysis showed that there were binding sites in the three-port-UTR region of microRNAs-34a and Tgif2. We used double luciferase reporter gene assay to determine whether Tgif2 is a direct target gene for microRNAs-34a. The fluorescence value of gf2-wild type luciferase reporter plasmid was significantly down-regulated (P 0.05), while the fluorescence value of Tigf2-mutant was not significantly different from that of Mimics (P 0.05), indicating that Mi-34a could bind to the three-port UTR seed region of target gene Tgif2 mRNA. Conclusion: 1. MiR-34a expression in peripheral blood of patients with hypertension was significantly increased, and was positively correlated with the clinical grade of hypertension; 2. MiR-34a could inhibit the proliferation and migration of HUVECs cells by down-regulating the expression of Tgif2 and promote their apoptosis. And promote vascular endothelial damage.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1
本文编号：2199314
[Abstract]:Background Essential hypertension is a common chronic disease and the most important risk factor of cardiovascular and cerebrovascular diseases. Its main complications, such as stroke, myocardial infarction, heart failure and chronic kidney disease, not only cause disability and high mortality, but also seriously consume medical and social resources to create family and society. In recent years, with the aging of the population, the improvement of living standards, the quickening pace of life and other factors, the incidence of essential hypertension continues to rise, and the proportion of young patients significantly increased, the prevention and treatment of essential hypertension caused researchers. Vascular injury is one of the basic pathological changes in hypertension, including vascular endothelial cell injury, smooth muscle cell proliferation and vascular remodeling. It has been found that vascular endothelial cell injury plays an important role in the development of hypertension. It can disrupt the homeostasis of NO and ET secretion and induce vasoconstriction, which increases circulatory resistance and promotes the development of hypertension. In hypertensive environments, the apoptosis rate of microvascular endothelial cells increases significantly, leading to a decrease in blood parallel pathways, an increase in peripheral circulation resistance, and an increase in blood pressure. MicroRNA (microRNA) is a kind of non-coding small RNA with a length of 18-24 nucleotides, which is highly conserved and mainly through three ports of target gene mRNA. Complete or incomplete binding of the untranslated region (3_-UTR) region to regulate gene expression at the posttranscriptional level by inhibiting protein translation or mRNA degradation. Current studies have shown that the expression profile of microRNAs in the peripheral blood of hypertensive patients has changed significantly, and even has high clinical value in early diagnosis and prognosis of hypertension. MiR-34a is a multifunctional regulator involved in the regulation of cell division, senescence, apoptosis and proliferation by regulating the expression of related target genes. In this study, we first examined the expression of microRNAs in the peripheral blood of patients with hypertension and its relationship with the clinicopathological characteristics of hypertension; then, we conducted in-depth studies in vitro to explore the role and mechanism of microRNAs in the injury of hypertensive endothelial cells. Objective To investigate the expression of microRNA-34a in peripheral blood of patients with hypertension and its molecular regulation mechanism in vascular endothelial cell injury induced by hypertension. MicroRNA-34a inhibitor and scramble-controlled microRNA were transfected into human umbilical vein endothelial cells (HUVEC) for further detection 48 hours. CCK-8, Transwell and flow cytometry were used to study the proliferation, cell cycle, apoptosis of HUVECs. The expression of Tgif2 in HUVECs was detected by Western blot. 6. The expression of Tgif2 in HUVECs was analyzed by SPSS16.0 statistical software. All measurements were expressed by mean (+) standard deviation (x (+) s). Results: 1. The expression of MiR-34a in the peripheral blood of patients with hypertension and its clinical significance Real-time fluorescence quantitative PCR results showed that compared with the normal control group, the expression of MiR-34a in the peripheral blood of patients with hypertension was significantly higher, the difference was statistically significant (P 0.05). The expression of microRNA-34a in peripheral blood of patients with stage 3 hypertension was significantly higher than that of patients with stage 1 and 2 hypertension (P MiR-34a may inhibit the proliferation of vascular endothelial cells in hypertensive patients. 3. MiR-34a may affect the migration of HUVECs. Transwell experiment showed that HUVECs migration ability was inhibited by down-regulating the expression of MiR-34a. The number of cells passing through the ventricular membrane was significantly higher than that of NC group (74.5+4.30 vs 48.5+6.3), and the difference was statistically significant (P The effect of MiR-34a on the cell cycle of HUVECs was detected by flow cytometry. The results showed that G1/S phase transition of HUVECs in the inhibitor group was faster than that in the NC group, indicating that overexpression of MiR-34a could regulate G1/S phase transition and inhibit the proliferation of HUVECs, thereby inhibiting the proliferation of HUVECs. Effect of MiR-34a on apoptosis of HUVECs Flow cytometry showed that the apoptosis rate of HUVECs decreased significantly after transfection of MiR-34a inhibitor (P 0.05), suggesting that reducing the expression of MiR-34a could inhibit the apoptosis of HUVECs during the development of hypertension, which may be induced by hypertension. Tigf2 is a target gene for microRNAs-34a. Bioinformatics analysis showed that there were binding sites in the three-port-UTR region of microRNAs-34a and Tgif2. We used double luciferase reporter gene assay to determine whether Tgif2 is a direct target gene for microRNAs-34a. The fluorescence value of gf2-wild type luciferase reporter plasmid was significantly down-regulated (P 0.05), while the fluorescence value of Tigf2-mutant was not significantly different from that of Mimics (P 0.05), indicating that Mi-34a could bind to the three-port UTR seed region of target gene Tgif2 mRNA. Conclusion: 1. MiR-34a expression in peripheral blood of patients with hypertension was significantly increased, and was positively correlated with the clinical grade of hypertension; 2. MiR-34a could inhibit the proliferation and migration of HUVECs cells by down-regulating the expression of Tgif2 and promote their apoptosis. And promote vascular endothelial damage.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1
【参考文献】
相关期刊论文 前3条
1 刘明波;李镒冲;刘世炜;王文;周脉耕;;2010年中国人群高血压疾病负担[J];中华流行病学杂志;2014年06期
2 范凡;孙爱军;卜丽萍;邹云增;葛均波;;miR34a通过调控乙醛脱氢酶2参与心肌细胞凋亡及心室重构[J];中国动脉硬化杂志;2011年03期
3 黎健;;miR-34a调控内皮祖细胞介导的血管新生[J];中国动脉硬化杂志;2011年03期
，本文编号：2199314
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2199314.html
最近更新
教材专著
