Bioengineering novel chimeric microrna-34a for prodrug cancer therapy: High-yield expression and purification, and structural and functional characterization

Wei Peng Wang, Pui Yan Ho, Qiu Xia Chen, Balasubrahmanyam Addepalli, Patrick A. Limbach, Mei Mei Li, Wen Juan Wu, Joseph L. Jilek, Jing Xin Qiu, Hong Jian Zhang, Tianhong Li, Theodore Wun, Ralph W deVere White, Kit Lam, Aiming Yu

Research output: Contribution to journalArticle

23 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)131-141
Number of pages11
JournalJournal of Pharmacology and Experimental Therapeutics
Volume354
Issue number2
DOIs
StatePublished - Aug 1 2015

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ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine

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