Li Mei-Mei, Addepalli Balasubrahmanyam, Tu Mei-Juan, Chen Qiu-Xia, Wang Wei-Peng, Limbach Patrick A, LaSalle Janine M, Zeng Su, Huang Min, Yu Ai-Ming
Department of Biochemistry & Molecular Medicine, University of California-Davis School of Medicine, Sacramento, California (M.-M.L., M.-J.T., Q.-X.C., W.-P.W., A.-M.Y.); Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China (M.-M.L, M.H.); Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (Q.-X.C., S.Z.); and Department of Medical Microbiology and Immunology, University of California Davis School of Medicine, Davis, California (J.M.L.).
Department of Biochemistry & Molecular Medicine, University of California-Davis School of Medicine, Sacramento, California (M.-M.L., M.-J.T., Q.-X.C., W.-P.W., A.-M.Y.); Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China (M.-M.L, M.H.); Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (Q.-X.C., S.Z.); and Department of Medical Microbiology and Immunology, University of California Davis School of Medicine, Davis, California (J.M.L.)
Drug Metab Dispos. 2015 Jul;43(7):1129-36. doi: 10.1124/dmd.115.064493. Epub 2015 May 1.
In contrast to the growing interests in studying noncoding RNAs (ncRNAs) such as microRNA (miRNA or miR) pharmacoepigenetics, there is a lack of efficient means to cost effectively produce large quantities of natural miRNA agents. Our recent efforts led to a successful production of chimeric pre-miR-27b in bacteria using a transfer RNA (tRNA)-based recombinant RNA technology, but at very low expression levels. Herein, we present a high-yield expression of chimeric pre-miR-1291 in common Escherichia coli strains using the same tRNA scaffold. The tRNA fusion pre-miR-1291 (tRNA/mir-1291) was then purified to high homogeneity using affinity chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes, including ABCC1, FOXA2, and MeCP2, as compared with cells transfected with the same doses of control methionyl-tRNA scaffold with a sephadex aptamer (tRNA/MSA). In addition, tRNA-carried pre-miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose-dependent manner, and significantly enhanced the sensitivity of ABCC1-overexpressing PANC-1 cells to doxorubicin. These results indicate that recombinant miR-1291 agent is effective in the modulation of target gene expression and chemosensitivity, which may provide insights into high-yield bioengineering of new ncRNA agents for pharmacoepigenetics research.
与对研究非编码RNA（ncRNA）如微小RNA（miRNA或miR）药物表观遗传学的兴趣日益增长形成对比的是，缺乏以经济有效的方式大量生产天然miRNA制剂的有效手段。我们最近的努力利用基于转运RNA（tRNA）的重组RNA技术在细菌中成功生产了嵌合前体miR-27b，但表达水平非常低。在此，我们展示了使用相同的tRNA支架在常见的大肠杆菌菌株中高效表达嵌合前体miR-1291。然后使用亲和色谱法将tRNA融合前体miR-1291（tRNA/mir-1291）纯化至高度均一，其一级序列和转录后修饰通过质谱分析直接表征。嵌合tRNA/mir-1291在人乳腺癌MCF-7和胰腺癌细胞中很容易加工成成熟的miR-1291。因此，与用相同剂量的带有葡聚糖凝胶适体的对照甲硫氨酰-tRNA支架（tRNA/MSA）转染的细胞相比，重组tRNA/mir-1291降低了miR-1291靶基因的蛋白质水平，包括ABCC1、FOXA2和MeCP2。此外，tRNA携带的前体miR-1291以剂量依赖性方式抑制MCF-7和胰腺癌细胞的生长，并显著增强过表达ABCC1的胰腺癌细胞对阿霉素的敏感性。这些结果表明重组miR-1291制剂在调节靶基因表达和化学敏感性方面是有效的，这可能为药物表观遗传学研究中新型ncRNA制剂的高产生物工程提供见解。
