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-miR34a (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 miRNAbased therapies.

MicroRNA Pharmacoepigenetics: Posttranscriptional Regulation Mechanisms behind Variable Drug Disposition and Strategy to Develop More Effective Therapy

“…However, research on miRNA pharmacoepigenetics and development of miRNA-based therapy may be limited by the utilization of synthetic miRNA agents consisting of excessive artificial modifications on the phosphate linkages and/or ribose rings and thus exhibit different physicochemical and biologic properties or toxicities. By contrast, recombinant RNA technology has been successfully employed to costeffectively produce large quantities of miRNA agents Chen et al, 2015;Li et al, 2015;Wang et al, 2015), which are comprised of no or posttranscriptional modifications on the nucleobases and may better capture the structures, functions, and safety properties of natural RNAs. These natural ncRNA agents are produced in cells and should represent a novel class of miRNA agents for research and development.…”

Bioengineered non-coding RNA agent (BERA) in action

“…Following the construction of target plasmid and transformation, target ncRNA is expressed in bacteria and purified to a high degree of homogeneity by anion exchange FPLC method. 40,43 We have further demonstrated that many chimeric ncRNAs (e.g., OnRS/miR-27b, OnRS/miR-124 and OnRS/GFP-siRNA, etc.) can be produced in E.coli on large scale, and be processed to target sRNA agents (e.g., miR-27b, miR-124 and GFP-siRNA, etc.)…”

“…Bioengineered tRNA/mir-34a agent indeed acted as a prodrug in suppressing tumor growth in both subcutaneous A549 xenograft 43 and orthotopic 143B xenograft 45 mouse models, while BERAs were well tolerated in mice. Combined with DNA or protein targeting agents, RNA targeting BERAs could have synergistic effects to combat lethal cancer, especially those lacking effective target therapeutics.…”

“…37,38 In addition, recombinant ncRNAs from, such as the siRNAs isolated from p19 complex and miRNAs/ siRNAs from OnRS platform (Table 1), are biologically active in the regulation of target gene expression in mammalian cells, which have been demonstrated in many studies. 22,34,35,40,43,44 Specifically, bioengineered tRNA/ mir-27b was found to be processed to mature miR-27b in human carcinoma cells, which consequently reduced CYP3A4 protein expression and led to a lower midazolam 1'-hydroxylase activity. 35 The tRNA/mir-1291 was readily processed to mature miR-1291 in breast cancer MCF-7 cells and pancreatic cancer PANC-1 cells.…”

“…34 Consequently, recombinant tRNA/mir-1291 reduced the protein levels of miR-1291 target genes and increased the sensitivity of carcinoma cells to chemotherapeutics. In addition, BERA miRNAs/siRNAs were more effective than synthetic miRNA/siRNA agents at same concentrations in the regulation of target gene expression and cell functions, 40,43 indicating that natural BERA agents could be valuable for functional and therapeutic studies.…”