Recombinant microRNAs in xenobiotic and nutrient disposition

项目总结 MicroRNAs(miRNAs或miRs)是基因组衍生的功能性非编码RNA(NcRNA)分子 管理细胞中靶基因表达的转录后调控。我们的长期目标是获得 MiRNA调控外源生物、营养物质分配和药物调控的机制研究 为开发新的治疗策略而采取的行动。我的实验室率先开展了关于 MiRNA药物表观遗传学为药物处置中的个体间差异提供了新的见解 和回应。由于患病的癌细胞沉迷于关键物质的持续供应和代谢 异种生物营养素(如氨基酸或氨基酸、维生素、糖等)对于不受控制的扩散和 肿瘤发生，了解代谢酶的作用(例如，吡哆醇-5-初级磷酸氧化酶 或PNPO)和转运体(例如AA转运体SLC7A5/LAT1、葡萄糖转运体SLC2A1/GLUT1等) 在营养代谢和运输方面的研究可能有助于确定新的治疗靶点。然而，有一个关键的差距。 在理解关键营养物质处置的重要调节因素和机制方面 和癌细胞的动态平衡。此外，目前对miRNA功能和治疗的研究包括 仅限于使用体外制造的化学工程miRNA“模拟物”，包括广泛的 各种类型的化学修饰，与天然RNA分子完全不同 在活细胞中产生和折叠，没有任何或只是携带有限数量的转录后 修改。这也与蛋白质研究和疗法形成鲜明对比，这些研究和疗法已经发现了终极 通过使用在活细胞中产生和折叠的重组或生物工程蛋白而不是 在体外化学合成的多肽或蛋白质。最近，我们建立了一部小说 基于tRNA/Pre-miRNA的RNA生物工程技术，可实现高产和大规模生产 通过体内细菌发酵获得重组miRNA试剂。我们的研究表明 重组miRNAs在调节人类细胞靶基因表达方面具有生物学活性，并且 随后调节药物处置和反应。此外，我们的初步研究表明， 人tRNA(HtRNA)可与人前miRNA(hsa-pre-miR)偶联为新型载体 用于生产完全人源化的重组或生物工程miRNA试剂，即 HBERA/miRNA。因此，在这一应用中，我们提出了(1)建立和利用新型的ncRNA载体 为了产生一组重组的hBERA/miRNA分子(目标1)，(2)描述 重组miRNAs在调控细胞维生素B6代谢和再生障碍性贫血中的作用 代谢组/内稳态(目标2)，以及(3)确定重组miRNAs在 体内疾病动物模型的药效学调节(目标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的机制作用 在控制异物/营养代谢以及它们在提高药物疗效方面的应用。

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.