Chimeric MicroRNA-1291 Biosynthesized Efficiently in Escherichia coli Is Effective to Reduce Target Gene Expression in Human Carcinoma Cells and Improve Chemosensitivity.
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.
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.).
Mei-Mei
MM
Li
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.).
Balasubrahmanyam
B
Addepalli
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.).
Mei-Juan
MJ
Tu
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.).
Qiu-Xia
QX
Chen
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.).
Wei-Peng
WP
Wang
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.).
Patrick A
PA
Limbach
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.).
Janine M
JM
LaSalle
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.).
Su
S
Zeng
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.).
Min
M
Huang
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.) aimyu@ucdavis.edu.
Ai-Ming
AM
Yu
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002277:Carcinoma / Q000378:metabolism*; D045744:Cell Line, Tumor; D004274:DNA, Recombinant / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism; D035683:MicroRNAs / Q000096:biosynthesis / Q000494:pharmacology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering
D000970:Antineoplastic Agents; D004274:DNA, Recombinant; D051542:Hepatocyte Nuclear Factor 3-beta; D051783:Methyl-CpG-Binding Protein 2; D035683:MicroRNAs; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C496502:FOXA2 protein, human; C497392:MECP2 protein, human; C587958:MIRN1291 microRNA, human; C421109:multidrug resistance-associated protein 1