miRNA Regulation of the Mitochondrial Genome

2型糖尿病的发病率急剧上升。危及生命的并发症之一是心脏 失败，在此之前是生物能量功能障碍。使用小鼠(db/db)和人类(患者)2型糖尿病 模型中，我们观察到明显的线粒体功能障碍，最终导致产生ATP的能力下降 用于心脏收缩。MicroRNAs(MiRs)是调节翻译的非编码RNA。使用交叉链接 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。 MIR移位到心肌线粒体内或移位出心肌线粒体的患者。特别令人感兴趣的是增加的mir- 378存在于线粒体基因组编码的ATP6信使核糖核酸的功能调节环境中 对于作为ATP合成酶复合体一部分的F0质子马达的一个亚单位。ATP合酶活性降低 功能性促进心脏中的生物能量缺陷，促进心力衰竭。尽管如此，目前 尚不清楚miR-378阻断是否可以减少与2型糖尿病相关的线粒体功能障碍 心脏通过与线粒体转录组的直接相互作用。此外，负责这一问题的机制 进入线粒体的miRs的动态流量是未知的。一种可能的机制涉及参与 我们观察到的线粒体RNA输入蛋白多核苷酸磷酸化酶(PNPase) 在db/db小鼠和2型糖尿病患者的线粒体中Be增加。拟议的研究旨在解决 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 以开始将这些发现转化为2型糖尿病患者。此应用程序的目标是 (1)检测miR-378缺失或功能抑制对2型糖尿病小鼠模型的体内疗效 恢复心肌线粒体ATP6蛋白表达和ATP生成能力；(2)评价 MiR-378抑制剂对2型糖尿病人心肌细胞的治疗作用 患者；以及(3)评估PNPase在推动miR-378通量进入 线粒体。这一应用的中心假设是，抑制miR-378将破坏其 翻译下调线粒体中的ATP6，保持ATP的生成能力并限制 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 通过操纵PNPase水平及其结构来调节。为了检验这一假设，一种创新的方法 已经提出，它使用了新的实验方法，在细胞，动物和 人体模型。建议的工作组合具有重要意义，因为它将提供对 调节线粒体miR分布的机制，同时提供对 抑制miR-378作为治疗策略的治疗潜力。我们的方法融合了机械性 对导致该型线粒体功能障碍的一条先前未探索的调控通路的检查 2糖尿病心脏临床前对参与轴的关键分子成分的评估。

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 presence 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 transcriptome. 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 begin to translate the findings to the type 2 diabetic patient. The objectives of this application are (1) determine the efficacy in vivo of miR-378 loss or its functional inhibition 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. The central hypothesis of this application is that inhibition of miR-378 will disrupt its ability to translationally down-regulate ATP6 in the mitochondrion, preserving ATP generating capacity and limiting cardiac contractile dysfunction in the type 2 diabetic heart. Further, miR-378 flux into the mitochondrion can be modulated by manipulating PNPase levels and its structure. To test this hypothesis, an innovative approach has been proposed which employs novel experimental methodologies that are tested in cellular, animal and human models. The combination of work proposed is significant because it will provide insight into the mechanisms regulating miR distribution in the mitochondrion while providing initial 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 type 2 diabetic heart with preclinical evaluation of key molecular constituents participating in the axis.