PO.MCB10.01 · 分子与细胞生物学
4-硫尿苷转录后修饰对生物工程改造的let-7e-5p在NSCLC细胞中抑癌活性的影响
The impact of 4-thiouridine posttranscriptional modification on the oncosuppressive activity of bioengineered let-7e-5p in NSCLC cells
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摘要 Abstract
中文摘要
目前的microRNA（miRNA）研究与治疗开发主要依赖体外化学合成的miRNA模拟物。尽管这类模拟物有效，但可能无法完全重现体内合成的天然miRNA分子的特性。本实验室开发了一种新型的基于tRNA融合pre-miRNA载体的RNA生物工程平台技术，能够通过体内细菌发酵一致、高产、大规模地生产生物型miRNA（BioRNA），在非小细胞肺癌（NSCLC）的实验性治疗中展现出强大潜力。在抑癌性let-7-5p异构体中，BioRNA/let-7e-5p被鉴定为抑制人NSCLC细胞活力最为有效的分子。此外，尽管我们观察到物种保守的转录后修饰，例如D环中的二氢尿苷（D）和2′-O-甲基鸟苷（Gm），以及T环中的5-甲基尿苷（m5U）和假尿苷（Y），但液相色谱-串联质谱（LC-MS/MS）和基于纳米孔的直接RNA测序研究揭示，在人tRNA片段的第8位（s4U8）存在一种细菌特异性的4-硫尿苷（s4U）修饰。为了对我们的BioRNA技术进行再创新并生成不含s4U修饰的BioRNA/let-7e-5p分子，我们采用了两种策略：（1）通过缺失或替换来改变U8；（2）在缺乏s4U合成能力的E. coli菌株中表达U8 BioRNA。两种方法均获得成功，使我们能够在每200 mL细菌培养物中生产出毫克级纯净、即用型的BioRNA/let-7e-5p分子，LC-MS/MS和纳米孔测序分析证实了对s4U修饰的规避。令人惊讶的是，全面的比较研究显示，远端s4U修饰对let-7e-5p从BioRNA中释放的影响极小，进而对NSCLC细胞中多个靶向癌基因的下调影响也极小。这些发现将指导BioRNA/let-7e-5p作为NSCLC治疗潜在疗法的开发。
查看英文原文 English abstract
Current microRNA (miRNA) research and therapeutic development primarily rely on miRNA mimics chemically synthesized in vitro . While effective, miRNA mimics may not fully recapitulate the properties of natural miRNA agents synthesized in vivo . Our lab has developed a novel tRNA-fused pre-miRNA carrier-based RNA bioengineering platform technology, enabling consistent, high-yield, and large-scale production of biologic miRNAs (BioRNAs) through in vivo bacterial fermentation, which have shown strong potential in experimental therapeutics for non-small cell lung cancer (NSCLC). Among the tumor suppressive let-7-5p isoforms, BioRNA/let-7e-5p was identified as the most effective to suppress human NSCLC cell viability. Further, while we observed species-conserved posttranscriptional modifications, such as dihydrouridine (D) and 2′- O -methylguanosine (Gm) in the D-loop as well as 5-methyluridine (m 5 U) and pseudouridine (Y) in the T-loop, liquid chromatography tandem mass spectrometry (LC-MS/MS) and nanopore-based direct RNA sequencing studies revealed the presence of a bacteria-specific 4-thiouridine (s 4 U) modification at position 8 (s 4 U8) of the human tRNA segment. To re-innovate our BioRNA technology and generate BioRNA/let-7e-5p molecules free of s 4 U modification, we employed two strategies: (1) change of U8 through deletion or substitutions, and (2) expression of U8 BioRNAs in an E. coli strain deficient in s 4 U synthesis. Both approaches proved successful, allowing us to produce milligrams of pure, ready-to-use BioRNA/let-7e-5p molecules per 200 mL bacterial culture, and LC-MS/MS and nanopore sequencing analyses confirmed the escape from s 4 U modification. Surprisingly, comprehensive comparative studies showed minimal effects of the distal s 4 U modification on the release of let-7e-5p from BioRNAs and consequently, downregulation of multiple targeted oncogenes in NSCLC cells. These findings will guide the development of BioRNA/let-7e-5p as potential therapeutics for NSCLC treatment.
利益披露 Disclosure
K. K. Wang, None..
M. Tu, None..
Y. Zhou, None..
Z. Wang, None..
P. A. Limbach, None..
H. Ding, None..
A. Yu, None.