title: Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells.
abstract: Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the regulation of various cellular processes, including drug metabolism and disposition. However, research on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre-miRNA-27b (miR-27b) agents can be biosynthesized in Escherichia coli using recombinant RNA technology, and recombinant transfer RNA (tRNA)/mir-27b chimera was readily purified to a high degree of homogeneity (>95%) using anion-exchange fast protein liquid chromatography. The tRNA-fusion miR-27b was revealed to be processed to mature miRNA miR-27b in human carcinoma LS-180 cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4), which consequently led to lower midazolam 1'-hydroxylase activity. These findings demonstrate that pre-miRNA agents can be produced by recombinant RNA technology for functional studies.
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, California (M.-M.L., W.-P.W., W.-J.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.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W.).
authors.forename: Mei-Mei
authors.initials: MM
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, California (M.-M.L., W.-P.W., W.-J.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.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W.).
authors.forename: Wei-Peng
authors.initials: WP
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, California (M.-M.L., W.-P.W., W.-J.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.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W.).
authors.forename: Wen-Juan
authors.initials: WJ
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, California (M.-M.L., W.-P.W., W.-J.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.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W.).
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, California (M.-M.L., W.-P.W., W.-J.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.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W.) aimyu@ucdavis.edu.
authors.forename: Ai-Ming
authors.initials: AM
authors.lastname: Yu
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D012313:RNA / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D017931:DNA Primers; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; D012313:RNA