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，这是由生物能量功能障碍之前。使用小鼠（db/db）和人（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 心脏收缩。微小RNA（miRs）是调节翻译的非编码RNA。使用交联 通过免疫沉淀和深度测序，我们在db/db和2型糖尿病患者中进行了令人兴奋的观察， miR在心肌线粒体内外易位的患者。特别感兴趣的是增加的miR- 378存在于线粒体基因组编码的ATP 6 mRNA的功能调控环境中， F0质子马达的一个亚基，是ATP合酶复合物的一部分。ATP合成酶减少 功能性的降低促进心脏中的生物能缺陷，从而促进心力衰竭。然而，目前 目前尚不清楚阻断miR-378是否能减少与2型糖尿病相关的线粒体功能障碍 通过与线粒体转录组的直接相互作用而影响心脏。此外，负责执行《公约》的机制 miR进入质子的动态通量是不确定的。一个潜在的机制涉及参与 线粒体RNA输入蛋白多核苷酸磷酸化酶（PNIPs），我们已经观察到， 在db/db小鼠和2型糖尿病患者的线粒体中增加。拟议的研究涉及 这些知识的差距，并将体外细胞方法与动物和人类实验模型相结合 努力开始将这些发现转化为2型糖尿病患者。本申请的目的是 (1)确定2型糖尿病小鼠模型中miR-378损失或其功能抑制的体内功效 用于恢复心脏中线粒体ATP 6蛋白表达和ATP产生能力;（2）评估 递送至来自2型糖尿病的分离的人心肌细胞的miR-378抑制剂的治疗功效 （3）评估PNTR对驱动miR-378通量进入PNTR的机制的贡献。 你好本申请的中心假设是miR-378的抑制将破坏其表达miR-378的能力。 抑制性下调ATP-6，保持ATP生成能力， 2型糖尿病心脏的心脏收缩功能障碍此外，miR-378进入细胞内的通量可以被调节。 通过操纵PNTR水平及其结构来调节。为了验证这一假设，一种创新的方法 已经提出了采用新的实验方法，在细胞，动物和 人体模型拟议的工作组合是重要的，因为它将提供对 调节miR在转录子中分布的机制，同时提供对miR在转录子中的表达的初步翻译见解。 miR-378抑制作为治疗策略的治疗潜力。我们的方法融合了 检查以前未探索的调节途径，有助于线粒体功能障碍的类型 2糖尿病心脏与参与轴的关键分子成分的临床前评价。

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.