miRNA Regulation of the Mitochondrial Genome

 描述(申请人提供)：2型糖尿病发病率急剧增加。在威胁生命的并发症中有心力衰竭，在此之前会出现生物能量障碍。使用小鼠(db/db)和人类(患者)2型糖尿病模型；我们观察到明显的线粒体功能障碍，最终导致为心脏收缩产生ATP的能力下降。MicroRNAs(MiRs)是调节翻译的非编码RNA。利用交联免疫沉淀和深度测序，我们在db/db和2型糖尿病患者中都进行了令人兴奋的观察，发现miRs移位到和移位出心肌线粒体。特别有趣的是，miR-378在功能调控背景下的存在增加，线粒体基因组编码的ATP6 mRNA编码F0质子马达的一个亚单位，该亚单位是ATP合成酶复合体的一部分。ATP合成酶功能降低会促进心脏中的生物能量缺乏，从而促进心力衰竭。尽管如此，它还是 目前尚不清楚miR-378阻断是否可以通过与线粒体基因组的直接相互作用来减少与2型糖尿病心脏相关的线粒体功能障碍。此外，负责miRs动态流入线粒体的机制尚不清楚。一种可能的机制涉及线粒体RNA导入蛋白多核苷酸磷酸化酶(PNPase)的参与，我们观察到在db/db小鼠和2型糖尿病患者的线粒体中PNPase增加。正在提出的研究解决了这些知识上的差距，并将体外细胞方法与动物和人类实验模型相结合，努力将这些发现转化为2型糖尿病患者。这一应用的中心假设是，抑制miR-378破坏了其下调线粒体中ATP6的能力，保留了ATP合成酶功能和ATP水平，并限制了2型糖尿病心脏的心脏收缩功能障碍。此外，miR-378进入线粒体的通量可以通过PNPase操作来调节。该应用的目的是：(1)确定预防性给药的miR-378抑制剂在体内的影响 该研究旨在：(1)在2型糖尿病小鼠模型中，用于恢复心肌线粒体ATP6蛋白的表达和ATP的生成能力；(2)评估miR-378抑制剂对2型糖尿病患者分离的人心肌细胞的治疗效果；(3)评估PNPase在推动miR-378流入线粒体的机制中的作用。为了验证这一假设，已经提出了一种创新的方法，使用Anagomir干预来操纵线粒体基因组编码的蛋白质，以努力缓解糖尿病相关的收缩功能障碍。所提出的工作组合具有重要意义，因为它将提供对调节线粒体中miR分布的机制的洞察，同时提供对miR-378抑制作为治疗策略的治疗潜力的翻译洞察。我们的方法结合了对以前未探索的导致糖尿病心脏线粒体功能障碍的调控途径的机械性检查，以及对参与轴的关键分子成分的临床前评估。

 DESCRIPTION (provided by applicant): Type 2 diabetes mellitus incidence has increased dramatically. Among the life threatening complications is heart failure, which is preceded by bioenergetic dysfunction. Using mouse (db/db) and human (patient) type 2 diabetic models; we observed pronounced mitochondrial dysfunction culminating in a decreased ability to generate ATP for cardiac contraction. MicroRNAs (miRs) are non-coding RNAs that regulate translation. Using cross-linking immunoprecipitation and deep sequencing, we made the exciting observation, in both db/db and type 2 diabetic patients that miRs translocate into and out of cardiac mitochondria. Of particular interest was an increased miR- 378 presences in a functional regulatory context with mitochondrial genome-encoded ATP6 mRNA which codes for a subunit of the F0 proton motor that is part of the ATP synthase complex. Decreased ATP synthase functionality promotes bioenergetic deficit in the heart, promoting heart failure. Nevertheless, it is currently unclear whether miR-378 blockade can reduce mitochondrial dysfunction associated with the type 2 diabetic heart by direct interaction with the mitochondrial genome. Further, the mechanisms responsible for the dynamic flux of miRs into the mitochondrion are undefined. One potential mechanism involves the participation of the mitochondrial RNA import protein polynucleotide phosphorylase (PNPase) which we have observed to be increased in mitochondria from db/db mice and type 2 diabetic patients. The studies being proposed address these gaps in knowledge and integrate in vitro cellular approaches with animal and human experimental models in an effort to translate the findings to the type 2 diabetic patient. The central hypothesis of this application is that inhibition of miR-378 disrupts its ability to down-regulate ATP6 in the mitochondrion, preserving ATP synthase function as well as ATP levels, and limiting cardiac contractile dysfunction in the type 2 diabetic hearts. Further, miR-378 flux into the mitochondrion can be modulated by PNPase manipulation. The objectives of this application are: (1) determine the impact in vivo of a prophylactically delivered miR-378 inhibitor in a type 2 diabetic mouse model for restoring mitochondrial ATP6 protein expression and ATP generating capacity in the heart; (2) evaluate the therapeutic efficacy of a miR-378 inhibitor delivered to isolated human cardiomyocytes from type 2 diabetic patients; and (3) assess the contribution of PNPase to the mechanisms driving miR-378 flux into the mitochondrion. To test this hypothesis, an innovative approach has been proposed which employs antagomir intervention to manipulate mitochondrial genome-encoded proteins in an effort to mitigate diabetes- associated contractile dysfunction. The combination of work proposed is significant because it will provide insight into the mechanisms regulating miR distribution in the mitochondrion while providing translational insight into the therapeutic potential of miR-378 inhibition as a treatment strategy. Our approach merges mechanistic examination of a previously unexplored regulatory pathway contributing to mitochondrial dysfunction in the diabetic heart with preclinical evaluation of key molecular constituents participating in the axis.