【作者】 杨盛兰；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2013， 博士

【摘要】 研究背景和目的：糖尿病（diabetes millitus, DM）是当今威胁人类健康的重大疾病。研究指出，现在全世界现有糖尿病患者2.85亿，预计到2030年，糖尿病的患病人数将几乎是现在的两倍。心血管并发症是糖尿病患者致残或致死的首要原因。其中，糖尿病独立于高血压、心脏瓣膜疾病、冠心病和及其他心脏病所导致的心脏结构和功能异常，即糖尿病心肌病（diabetic cardiomyopathy）越来越受到研究者重视。研究者们相继发现脂质毒性（lipotoxicity）、糖毒性、细胞内钙稳态紊乱、氧化应激增强、代谢底物转变、线粒体功能失常、炎症和内质网应激（endoplasmic reticulum stress, ER stress）等都是参与2型糖尿病心肌病的致病机制。其中脂质毒性对心脏功能和结构的损伤是糖尿病心肌病的发生发展的关键环节，但确切的分子机制还不甚明了。最近有研究指出，microRNA和自噬都参与胰岛素抵抗时心脏病变过程，为我们对糖尿病心肌病的分子机制研究提供了新的视角。微小RNA（microRNAs, miRNAs）是一类广泛存在于生物体内，22-23个碱基长度的非编码单链RNA。MiRNAs通过与靶mRNA的3’-非翻译区（3’-untranslated region,3’UTR）特异性结合，在转录后水平调控靶基因的表达。大量的研究表明miRNAs参与机体的各种生命进程；同时，miRNAs功能异常参与各种疾病过程。近年来的研究表明，miRNAs，尤其是心脏高丰度表达的miRNAs，在各种心脏病发病机制中扮演着重要作用。研究人员通过在心肌细胞过表达这些miRNAs，导致心肌细胞发生肥大或体积减小，揭示了miRNA在心肌肥厚、心室重构等病理生理过程中起着关键作用。然而miRNAs在糖尿病及其心血管并发症中的作用还不是很明确。自噬是一种对细胞内成分降解再利用的保守进化过程，通过对细胞内长寿命蛋白或损伤蛋白及细胞器的降解，自噬维持着细胞生存。正常情况下，心脏低水平自噬是一种细胞应激的保护机制；然而，自噬过度也会导致细胞损伤。最近有研究指出，在果糖诱导胰岛素抵抗的小鼠模型中，自噬的激活与心肌细胞过氧化产物增多、纤维化及细胞死亡有关，从而参与形成心脏胰岛素抵抗，提示自噬在糖尿病心肌病致病机制中扮演重要角色。此外最近关于miR-30a参与调控自噬的研究引起广泛关注，说明miRNAs在自噬的调控中起着重要作用。MiR-30家族是在心肌细胞高丰度表达的miRNAs之一。过去实验证明，miR-30参与调节心肌肥大、心室重塑、心肌纤维化等一系列心脏功能异常的致病过程。但是miR-30c在糖尿病时心脏中的作用还不清楚。此外最近关于miR-30a参与调控自噬的研究引起广泛关注，说明miRNAs在自噬的调控中起着重要作用。miR-30c、自噬及糖尿病心肌病之间的调节关系启发我们探究：miR-30c是否参与了2型糖尿病的病理过程？如果参与，miR-30c是否是通过调节自噬参与了糖尿病心肌病致病过程？因此，我们围绕假设，展开相关研究，试图从miRNAs的角度寻找新的糖尿病心肌病的发病机制和治疗方案。实验方法：运用real-time PCR对2型糖尿病db/db小鼠心肌和正常对照C57BL/Ks小鼠心肌分别进行检测，从中了解miR-30c是否存在差异型表达。利用western-blot技术，在蛋白水平检测自噬强度在db/db小鼠心肌中相对于正常对照C57BL/Ks小鼠心肌是否有所改变。用高浓度FFAs培养H9c2心肌细胞，在细胞实验中检测miR-30c及自噬发生受FFAs调控。利用生物信息学软件预测miR-30c的作用靶点，将挑选出的预测的靶mRNA的3’UTR区克隆至pMIR-Report载体，运用荧光报告基因技术验证靶点，并利用western-blot技术再次验证靶点。利用我们特有的心肌特异性病毒表达载体recombinant adeno-associated virusvector9（rAAV9），将miR-30c、anti-miR-30c及靶mRNA分别包装至病毒表达载体，并检测滴度及转染效率。将上述rAAV载体介导体内表达，在db/db小鼠心肌中差异性表达miR-30c，并将靶mRNA回复，验证miR-30c的载体沉默效应。通过小鼠心脏超声和血流动力学分析检测差异表达miR-30c是否影响小鼠心脏功能同时western-blot和电镜检测差异表达miR-30c对自噬通路的影响。利用生物信息学软件预测miR-30c启动子区的上游调控靶点，miR-30c的5’-promoter区克隆至pGL3-Report载体，运用荧光报告基因技术验证靶点，并利用western-blot技术再次验证上游调控。实验结果：1.2型糖尿病小鼠心肌组织中差异性表达miR-30c通过real-time PCR检测证实了miR-30c的差异性表达。验证相对于正常对照，在2型糖尿病心肌病中miR-30c表达下调。2.2型糖尿病小鼠心肌组织中自噬增强利用western-blot检测自噬相关蛋白Beclin-1、LC3-Ⅱ蛋白表达，证明相对于正常对照，2型糖尿病小鼠心肌中自噬增强。3.高浓度FFAs培养H9c2心肌细胞miR-30c下调、自噬发生增强高水平FFAs培养心肌细胞H9c2，相对于未处理对照组，real-time PCR检测表明miR-30c明显下调；western-blot证明FFAs诱导自噬增强4. miR-30c靶点预测及验证利用TARGETSCAN预测分析网站对miR-30c的作用靶mRNAs进行预测分析，筛选出Beclin-1是miR-30c可能的靶点mRNA，将其3’UTR区序列构建至pMIR-Report载体，通过荧光报告基因技术结果提示Belcin-1为miR-30c的作用靶基因，同时采用western-blot进行了蛋白水平验证。5. rAAV-miR-30c、rAAV-anti-miR-30c及rAAV-Beclin-1病毒包装及rAAV载体介导miR-30c体内差异性表达及其对2型糖尿病心脏功能调节分析及对自噬的影响采用rAAV9包装系统，分别包装rAAV-miR-30c、rAAV-anti-miR-30c及rAAV-Beclin-1病毒，real-time PCR检测病毒滴度，同时转染rAAV-GFP检测转染效率。将db/db小鼠尾静脉分别注射rAAV-miR-30c、rAAV-anti-miR-30c，使miR-30c在心肌中差异性表达。并另外注射rAAV-Beclin-1病毒逆转过表达miR-30c对Beclin-1的抑制效应。小鼠心脏超声和血流动力学检测发现，相对于对照组，rAAV-miR-30c组心功能增强。Western-blot发现过表达miR-30c使Beclin-1、LC3-Ⅱ蛋白水平降低，提示自噬降低，并通过电镜进一步验证过表达rAAV-miR-30c使自噬小体减少。但rAAV-Beclin-1回复后，逆转了miR-30c的效应，自噬减弱。同时，rAAV-anti-miR-30c组心功能受损，自噬增强。6. MiR-30c上游表达调控利用生物信息学软件预测出NF-κB调控miR-30c启动子区，miR-30c的5’-promoter区克隆至pGL3-Report载体，运用报告基因技术验证NF-κB对miR-30c启动子区的调控，并利用real-time PCR检测再次验证NF-κB上游调控miR-30c表达。7.db/db小鼠心脏中NF-κB表达研究通过对db/db小鼠心脏组织的免疫组化检测，发现NF-κB表达降低；同时其抑制分子IκB-α表达增强。结论：本实验通过检测2型糖尿病心肌病动物模型db/db小鼠心肌组织中的差异性表达miR-30c，利用分子生物学、细胞生物学技术及生物信息学技术，对miR-30c在体内、体外对糖尿病时心肌细胞的作用及其机制进行了研究，得出以下结论：1.2型糖尿病小鼠db/db心肌细胞中或高浓度FFAs诱导心肌细胞时，miR-30c表达下调，自噬增强；2. miR-30c能通过沉默Beclin-1，抑制自噬，从而保护糖尿病时心脏功能；3. NF-κB是miR-30c启动子区重要的正向调控因子，并在db/db小鼠糖尿病发展的中晚期表达下调。综合结论：2型糖尿病时，受NF-κB调控的miR-30c表达减少，通过其靶基因Beclin-1负性调控诱发过度自噬，参与心肌病的致病过程。该结论为糖尿病心肌病寻找新的基因靶向治疗方法提供了线索。更多还原

【Abstract】 BackgroundDiabetes millitus (DM) has been a serious disease for peoples’ lives. It is indicatedthat there are285million diabetes patients in the world, and the number of diabetes willalmost doubles in2025. Cardiovascular complications are the leading cause of mortality indiabetic patients. Among them, diabetic cardiomyopathy exhibites abnormal cardiacstructures and functions, which is independent of hypertension, heart valve abnormalities,coronary diseases and other heart diseases. Emerging researches have showed thatlipotoxicity, glucotoxicity, impaired calcium homeostasis, altered metabolic substrates,oxidative stress, mitochondrial dysfunction, inflammation, and endoplasmic reticulumstress (ER stress) are all involved in pathogenic process of type2diabetic cardiomyopathy,but the exact molecular mechanism is still unclear. Recent studies have reported thatmicroRNAs and autophagy are associated with insulin resistance in myocardium, whichinspires us from a new perspective.MicroRNAs (miRNAs, miR) is a class of non-coding small RNAs widely existing inthe organism, uaually22~23-nucleotide single-stranded. Through pair-binding with 3’-untranslated region (3’-UTR) of target mRNA, miRNAs regulate the expression of targetgenes in the post-transcription level. Many experiments have proved that miRNAsparticipate in serious biological processes; at the same time, dysfunctions of miRNAs areinvoled in a variety of diseases. Recent researches have shown that miRNAs, especiallycardiac-abundant miRNAs, play important roles in the pathogenesis of many heart diseases.The researchers over-expressed these miRNAs in myocardial cells, and they becamehypertrophy or volume was decreased, revealling the crucial roles of miRNAs in thepathological process of myocardial hypertrophy and ventricular remodeling. However, therole of miRNAs in diabetes and the cardiovascular complications is still unknown.Autophagy is a conserved procress which degradates intracellular components forreuse. By degradation of long-life proteins, impired proteins and damaged organelles,autophagy serves for cell survivals. Under basal circumstances, low level autophagy in theheart is a protect mechanism response to stress; however, excessive autophagy may lead toheart cell damages. Recent studies have pointed out that in the fructose-induced mice model,the activation of autophagy is associated with increased cardiac peroxidation products,fibrosis, and cell death, therefore participating in the formation of insulin resistance in theheart. It suggests autophagy has a key role in the pathogenic mechanism of diabeticcardiomyopathy. In addition, a recent report that miR-30a regulates autophagy in cancercells attracts widely attentions, indicating miRNAs play an important role in the regulationof autophagy.MiR-30family is one of the cardiac high-expresed miRNAs. Earlier experimentsshowed that miR-30participated in the regulation of myocardial hypertrophy, ventricularremodeling, myocardial fibrosis and a series of cardiac dysfunctions. But the role ofmiR-30c in diabetic heart is unclear. The regululating relationship among MiR-30c,autophagy and diabetic cardiomyopathy inspires us to wonder: whether miR-30c isinvolved in the pathological process of type2diabetes? If this is the case, whether miR-30cparticipates in the pathogenic process of diabetic cardiomyopathy through regulation of autophagy? Focusing on these hypothesizes, we explore to find a novel therauputic way fordiabetic cardiomyopathy from miRNAs.MethodsUse real-time PCR to test if there are differences in cardiac expression of miR-30cbetween type2diabetes db/db mice and normal controls C57BL/Ks mice.Western-blot was employ to detect in protein level that whether autophagy wasdifferently regulated in myocardium of db/db mice compared with C57BL/Ks controls.H9c2cardiac cells were cultured in high concentrations of FFAs to test expression ofmiR-30c and autophagy in stimulation of FFAs.Use bioinformatic software to predict the potentcial targets of miR-30c. Then clone the3’-UTR of potential target mRNA to pMIR-Report vector, and verify the target gene ofmiR-30c by luciferase report assay, Western-blot was used to re-verify the target gene.Package miR-30c, anti-miR-30c and target mRNA respectively to cardiac-specificviral expression vector, recombinant adeno-associated virus vector9(rAAV9), and detectvirus titer and transfection efficiency.Express the relative viruses above in myocardium of db/db mice by tail vein injection.Employ echocardiographic test and hemodynamic analysis to examine cardiacfunction of treated db/db mice.Western-blot and electron micrograghic analysis are used to test whether autophagyflux is regulated by differentially expressed miR-30c.Bioinformatic software is utilized to predict upstream regulator of miR-30c. Clone5’-promoter region of miR-30c to pGL3report vector, and testify the up-stream regulator byluciferase report assay. Re-verify the regulation by western-blot. Results1. Differential expression of miR-30c in type2diabetic myocardial tissue comparedwith controlThrough the real-time PCR detection, miR-30c expression in db/db mice cardium isconfirmed to be decreased compared with normal controls.2. Enhanced Autophagy in myocardial tissue of type2diabetes.Beclin1and LC3-Ⅱ, autophagy associated proteins, are increased in db/db heartscompared with controls, suggesting autophagy flux is enhanced.3. Regulaltion of FFAs in vitroExpression of miR-30c was down-regulated while Beclin1and LC3-Ⅱ protein levelswere increased in H9c2cultured in high concentration of FFAs compared with controls,suggesting FFAs stimulates autophagy in heart cells.4. Target gene of miR-30cBeclin1was predicted to be the most putative target gene of miR-30c byTARGETSCAN software. Luciferase report assay and western-blot comfired miR-30ccould silenced expression of Beclin-1.5. Regulation of miR-30c in db/db heartEchocardiographic test and hemodynamic analysis showed overexpression of miR-30cresulted in alleviation of cardiac dysfunction in db/db mice, which could be encountered byco-overexpression of Beclin-1; while knockdown of miR-30c deteriorating the cardiacdysfunction. At the same time, western-blot and electron micrograghic analysis indicatedthat overexpression of miR-30c inhibited the beclin-1promoted autophagy in db/db hearts,but anti-miR-30c enhanced excessive autophagy. 6. Upstream regulator of miR-30cThrough bioinformatic prediction, NF-κB was identified as the putative regulator for5’-promoter region of miR-30c. Luciferase report assay and real-time PCR confirmed thatNF-κB enhanced miR-30c expression by regulating the5’-promoter region of miR-30c.7. Repression of NF-κB in late stage of type2diabetesIn the heart of late stage of type2diabetic db/db, expression of NF-κB was reducedwhile its antagonist IκB-α was enhanced.ConclusionIn our study, cardiac tissues from type2diabetic model db/db mice and normalcontrol C57BL/Ks were explored. Using molecular biology, cell biology and bioinformaticstechnology, the functions and mechanisms of miRNA-30c in diabetic heart wereinvestigated in vivo and in vitro. We found that:1. Expression of miR-30c is decreased while autophagy is enhanced in the heart ofdb/db mice or FFAs treated H9c2cells;2. miR-30c can inhibit autophagy by repression of Beclin-1, therefore serves as aprotective regulator in diabetic heart;3. NF-κB positively regulates expression of miR-30c, and down-regulated NF-κBcontributes to loss of miR-30c in late stage of type2diabetic db/db mice.Conclusively, in type2diabetes, loss of miR-30c contributes to the process of diabeticcardiomyopathy through enhancement of Beclin-1promoted autophagy. This result mayprovide a novel therapeutic way for diabetic heart from a new point of view.更多还原