A novel mouse model to distinguish the specific physiological significance of RNAi and biophysical mechanisms of microRNA

项目摘要 microRNA（miRs）是一类进化上保守的非编码小RNA分子，控制着大部分生物学功能， 事件，包括凋亡、细胞增殖、代谢、细胞命运决定、器官发生、发育， 应激反应和肿瘤发生。传统上，已知miR负调控基因表达 RNA干扰（RNAi）机制。最近，我们揭示了miR 1的一种新的生物物理作用， 是心脏中最主要的miR，在人心力衰竭中下调。我们发现 miR 1直接与内向整流钾通道Kir2.1结合，导致IK 1的直接抑制 电流并导致心肌细胞电生理功能的生物物理调节。我们 研究表明，miR 1通过两个途径调节组织/器官的发育和稳态， 不同的机制：新发现的生物物理调制的即时效应（秒到分钟） 以及RNAi的长期效应（数小时至数天）。有了这一重要的新发现， 了解这两种不同的miR作用机制如何协调以调节发育， 我们身体的内稳态。然而，没有有效的模型可以区分特定的生理 生物物理调节对RNAi机制的意义。我们发现一种与疟疾有关的 hSNP 14 A/G特异性地缺陷生物物理作用，同时维持miR 1的RNAi功能;因此，我们 我建议开发一种独特的转基因小鼠模型，可以将来自 生物物理调节和解剖RNAi在维持多细胞内稳态中的纯贡献， 器官/系统。我们将开发miR 1-全KO/肌肉特异性诱导型hSNP 14 A/G-敲入小鼠， 假设hSNP 14 A/G在肌肉细胞中的表达可以挽救miR 1-full- KO小鼠。我们将研究缺乏hSNP 14 A/G的生物物理功能是否会导致任何异常表型 (Aim 1），如心律失常，心力衰竭和骨骼肌收缩力异常，这将证明 miR 1的生物物理调节在体内稳态调节中的特定作用。我们还将关闭 hSNP 14 A/G的表达，并研究其特异性生理学特性。 miR 1的RNAi机制在心脏中的重要性（目的2）。这种独特的动物模型将非常有价值， 研究miR 1在多个器官/系统中的关键作用，包括心脏，骨骼肌，各种 癌症的类型。了解miR的生物物理调节和RNAi在体内的具体贡献 将扩大miRs的生物学意义，并指导我们开发新的治疗方法， 通过靶向miR的疾病。

Project Summary MicroRNAs (miRs) are evolutionally conserved small non-coding RNA molecules and control most biological events, including apoptosis, cell proliferation, metabolism, cell fate determination, organogenesis, development, stress responses, and tumorigenesis. Classically, miRs are known to negatively regulate gene expression through RNA interference (RNAi) mechanism. Recently, we revealed a novel biophysical action of miR1, which is the most predominant miR in the heart and is downregulated in human heart failure. We discovered that miR1directly binds to inward rectifier potassium channel Kir2.1, resulting in direct suppression of the IK1 current and leading to biophysical modulation of cardiomyocyte cellular electrophysiological functions. Our studies suggest that miR1 modulates the development and homeostasis of tissues/organs through two different mechanisms: the immediate effect (seconds to minutes) of newly-discovered biophysical modulation and long-term effect (hours to days) of RNAi. With this important new finding, it now becomes essential to understand how these two distinct miRs mechanisms of action coordinate to regulate the development and homeostasis of our body. However, there is no valid model that can distinguish the specific physiology significance of biophysical modulation versus RNAi mechanism. We found that an arrhythmia-associated hSNP14A/G specifically defects the biophysical action while maintaining miR1’s RNAi function; therefore, we propose to develop a unique transgenic mouse model that can separate the specific contribution coming from the biophysical modulation and dissect the pure contribution of RNAi in maintain the homeostasis of multi organs/systems. We will develop miR1-full-KO/muscle-specific inducible hSNP14A/G-knock-in mice, and we hypothesize that an expression of hSNP14A/G in muscle cells could rescue the postnatal lethality of miR1-full- KO mice. We will investigate if lacking biophysical function of hSNP14A/G induces any abnormal phenotypes (Aim 1), such as arrhythmia, heart failure, and abnormal contractility of skeletal muscle, which will demonstrate the specific role of miR1’s biophysical modulation in regulation of the homeostasis in vivo. We will also turn off the expression of hSNP14A/G by administration of doxycycline and investigate the specific physiological significance of miR1’s RNAi mechanism in the heart (Aim 2). This unique animal model will be very valuable to investigate the critical role of miR1 in multiple organs/systems, including the heart, skeletal muscle, various types of cancers. Understanding the specific contributions of miR’s biophysical modulation and RNAi in vivo will expand the biological significance of miRs and guide us to develop new therapeutic approaches for human diseases through targeting of miRs.