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

项目摘要 MicroRNAs(MiRs)是进化上保守的非编码小RNA分子，控制着大多数生物 包括细胞凋亡、细胞增殖、新陈代谢、细胞命运决定、器官发生、发育、 应激反应和肿瘤发生。经典地说，miRs被认为是负向调节基因表达的。 通过RNA干扰(RNAi)机制。最近，我们揭示了miR1的一种新的生物物理作用，它 是心脏中最主要的MIR，在人类心力衰竭中下调。我们发现 MiR1直接与内向整流钾通道Kir2.1结合，导致IK1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制。我们的 研究表明miR1通过两种途径调节组织/器官的发育和动态平衡 不同的机制：新发现的生物物理调制的即时影响(几秒钟到几分钟) 和RNAi的长期效应(几小时到几天)。有了这一重要的新发现，现在变得至关重要 了解这两种不同的MIR行动机制是如何协调的，以调节发展和 我们身体的动态平衡。然而，目前还没有一个有效的模型来区分特定的生理 生物物理调控与RNAi机制的意义。我们发现一种与心律失常相关的 HSNP14A/G在维持miR1‘S RNAi功能的同时，特异性地缺陷了生物物理作用；因此，我们 建议开发一种独特的转基因小鼠模型，可以分离来自 RNAi的生物物理调控及其在维持多细胞动态平衡中的纯作用 器官/系统。我们将开发miR1-Full-KO/肌肉特异性诱导型hSNP14A/G敲入小鼠，我们 假设hSNP14A/G在肌肉细胞中的表达可以挽救miR1-Full的出生后致命性. Ko老鼠。我们将调查hSNP14A/G缺乏生物物理功能是否会导致任何异常表型 (目标1)，如心律失常、心力衰竭和骨骼肌异常收缩，这将证明 MiR1‘S生物物理调控在体内动态平衡调节中的具体作用。我们还将关闭 多西环素对hSNP14A/G基因的表达及其特异性生理作用的研究 MiR1‘S RNAi机制在心脏中的意义(目标2)。这种独特的动物模型将对 研究miR1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的生物物理调控和RNAi在体内的具体作用 将扩大MIR的生物学意义，并指导我们开发新的人类治疗方法 通过靶向MIR治疗疾病。

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.