title: Bioengineering Novel Chimeric microRNA-34a for Prodrug Cancer Therapy: High-Yield Expression and Purification, and Structural and Functional Characterization.
abstract: Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Wei-Peng
authors.initials: WP
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Pui Yan
authors.initials: PY
authors.lastname: Ho
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Qiu-Xia
authors.initials: QX
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Balasubrahmanyam
authors.initials: B
authors.lastname: Addepalli
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Limbach
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Mei-Mei
authors.initials: MM
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Wen-Juan
authors.initials: WJ
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Jilek
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Jing-Xin
authors.initials: JX
authors.lastname: Qiu
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Hong-Jian
authors.initials: HJ
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Tianhong
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Theodore
authors.initials: T
authors.lastname: Wun
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Ralph DeVere
authors.initials: RD
authors.lastname: White
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.).
authors.forename: Kit S
authors.initials: KS
authors.lastname: Lam
authors.affiliation: Department of Biochemistry and Molecular Medicine (W.-P.W., P.Y.H., Q.-X.C., M.-M.L., W.-J.W., J.L.J., K.S.L., A.-M.Y.), Division of Hematology Oncology (T.L., T.W., K.S.L.) and Department of Urology (R.D.W.), School of Medicine, University of California-Davis, Sacramento, California; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio (B.A., P.A.L.); Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (J.-X.Q.); and Center of Drug Metabolism and Pharmacokinetics, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu, China (W.-P.W., H.-J.Z.) aimyu@ucdavis.edu.
authors.forename: Ai-Ming
authors.initials: AM
authors.lastname: Yu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D057005:Bioengineering / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D011355:Prodrugs / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D013329:Structure-Activity Relationship; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D000970:Antineoplastic Agents; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D011355:Prodrugs