RT Journal Article
SR Electronic
T1 Bioengineering novel chimeric microRNA-34a for prodrug cancer therapy: High-yield expression and purification, and structural and functional characterization
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP jpet.115.225631
DO 10.1124/jpet.115.225631
A1 Wei-Peng Wang
A1 Pui Yan Ho
A1 Qiu-Xia Chen
A1 Balasubrahmanyam Addepalli
A1 Patrick A. Limbach
A1 Mei-Mei Li
A1 Wen-Juan Wu
A1 Joseph L. Jilek
A1 Jing-Xin Qiu
A1 Hong-Jian Zhang
A1 Tianhong Li
A1 Theodore Wun
A1 Ralph DeVere White
A1 Kit S. Lam
A1 Ai-Ming Yu
YR 2015
UL http://jpet.aspetjournals.org/content/early/2015/05/28/jpet.115.225631.abstract
AB Development of microRNA (miRNA or miR) based treatments such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein we present a new approach to high-yield and large-scale biosynthesis of chimeric miR-34a agent in Escherichia coli using tRNA scaffold, which may act as a prodrug for cancer 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 (FPLC), whose primary sequence and posttranscriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and qPCR studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, whereas 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 varoius types of human carcinoma cells in a dose dependent manner and to much greater degrees 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 to the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effects on blood chemistries and IL-6 levels in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biological miRNAs to perform miRNA actions, and point towards a new direction to develop miRNA-based therapies.