Recombinant microRNAs in xenobiotic and nutrient disposition

项目摘要 microRNA（miRNAs或miRs）是基因组衍生的功能性非编码RNA（ncRNA）分子， 控制细胞中靶基因表达的转录后调节。我们的长远目标是 对miRNA控制的外源性物质和营养物处置的调节机制的理解和药物 为发展新的治疗策略而采取的行动。我的实验室率先研究了 miRNA药物表观遗传学为药物处置的个体间变异性提供了新的见解 和回应。由于病变的癌细胞依赖于持续的供应和代谢的关键 异生素营养物（例如，氨基酸或AA、维生素、糖等）不受控制的扩散， 肿瘤发生，了解代谢酶的作用（例如，吡哆醇-5-伯磷酸氧化酶 或PNPO）和转运体（例如，AA转运蛋白SLC 7A 5/LAT 1、葡萄糖转运蛋白SLC 2A 1/GLUT 1等） 在营养代谢和运输中的作用可能有助于确定新的治疗靶点。然而， 在理解重要的调控因素和机制的基础上， 和癌细胞内的稳态。此外，目前关于miRNA功能和治疗的研究是 局限于使用体外制备的化学工程化的miRNA“模拟物”， 各种类型的化学修饰，这是完全不同的天然RNA分子 在活细胞中产生和折叠，没有任何或仅携带有限数量的转录后 修改.这也与蛋白质研究和治疗形成鲜明对比， 通过使用在活细胞中产生和折叠的重组或生物工程蛋白质，而不是 在体外化学合成的多肽或蛋白质。最近，我们创作了一部小说 基于tRNA/pre-miRNA的RNA生物工程技术，可实现高产量和大规模生产 的重组miRNA试剂通过体内细菌发酵。我们的研究表明， 重组miRNA在调节人细胞中的靶基因表达方面具有生物活性，并且 随后调节药物处置和反应。此外，我们的初步研究显示， 特定的人tRNA（htRNA）可以与人pre-miRNA（hsa-pre-miR）偶联作为新的载体， 用于生产完全人源化的重组或生物工程化的miRNA试剂，即 hBERA/miRNA。因此，在本申请中，我们提出（1）建立和利用新型ncRNA载体 为了产生重组hBERA/miRNA分子的集合（目的1），（2）描绘了 重组miRNAs在细胞维生素B6代谢和AA调控中的作用 代谢组学/稳态（目的2），和（3）定义重组miRNA在代谢组学/稳态中的有效性。 体内疾病动物模型中药效学的调节（目的3）。拟议的研究将 建立一种独一无二的RNA生物技术，并确定几种人类miRNAs的作用机制 控制异生素/营养代谢以及它们在提高药物功效方面的应用。

PROJECT SUMMARY MicroRNAs (miRNAs or miRs) are genome-derived, functional noncoding RNA (ncRNA) molecules that govern posttranscriptional regulation of target gene expression in cells. Our long-term objective is to gain a mechanistic understanding of miRNA-controlled regulation of xenobiotic & nutrient disposition and drug actions towards the development of new therapeutic strategies. My laboratory pioneered the research on miRNA pharmacoepigenetics that has offered new insights into inter-individual variability in drug disposition and response. Since the diseased carcinoma cells are addicted to continuous supply and metabolism of key xenobiotic nutrients (e.g., amino acids or AAs, vitamins, sugars, etc.) for uncontrolled proliferation and tumorigenesis, understanding the roles of metabolic enzymes (e.g., pyridoxine-5-prime-phosphate oxidase or PNPO) and transporters (e.g., AA transporter SLC7A5/LAT1, glucose transporter SLC2A1/GLUT1, etc.) in nutrient metabolism and transport may help to identify new therapeutic targets. Yet, there is a critical gap in the understanding of important regulatory factors and mechanisms underlying key nutrients’ disposition and homeostasis in carcinoma cells. In addition, current research on miRNA functions and therapeutics are limited to the use of chemo-engineered miRNA “mimics” made in vitro and comprised of extensive and various types of chemical modifications, which are completely different from natural RNA molecules produced and folded in living cells without any or just carrying a limited number of posttranscriptional modifications. This is also in sharp contrast to protein research and therapy that have found ultimate success by using recombinant or bioengineered proteins produced and folded in living cells, rather than polypeptides or proteins synthesized chemically in vitro. Very recently, we have established a novel tRNA/pre-miRNA-based RNA bioengineering technology that permits high-yield and large-scale production of recombinant miRNA agents through in vivo bacterial fermentation. Our studies have showed that recombinant miRNAs are biologically active in regulating target gene expression in human cells, and subsequently modulate drug disposition and response. Furthermore, our preliminary studies have revealed that particular human tRNA (htRNA) can be coupled with human pre-miRNA (hsa-pre-miR) as novel carriers for the production of fully-humanized recombinant or bioengineered miRNA agents, namely hBERA/miRNA. Therefore, in this application, we proposed to (1) establish and utilize novel ncRNA carriers to produce a collection of recombinant hBERA/miRNA molecules (Aim 1), (2) delineate the mechanistic actions of recombinant miRNAs in the control of cellular vitamin B6 metabolism and AA metabolome/homeostasis (Aim 2), and (3) define the effectiveness of recombinant miRNAs in the modulation of pharmacodynamics in disease animal models in vivo (Aim 3). The proposed research will establish a one-of-a-kind RNA biotechnology, and define the mechanistic actions of several human miRNAs in the control of xenobiotic/nutrient metabolism as well as their applications to improving drug efficacy.