A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

Huiling Qiu; Jiasheng Zhong; Lan Luo; Nian Liu; Kang Kang; Junle Qu; Wenda Peng; Deming Gou

doi:10.1371/journal.pone.0143864

A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition

PLoS One. 2015 Dec 1;10(12):e0143864. doi: 10.1371/journal.pone.0143864. eCollection 2015.

Authors

Huiling Qiu^{1

2}, Jiasheng Zhong¹, Lan Luo¹, Nian Liu¹, Kang Kang³, Junle Qu², Wenda Peng², Deming Gou¹

Affiliations

¹ Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
² Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.
³ Department of Physiology, Shenzhen University Health Science Center, Shenzhen, Guangdong, 518000, China.

Abstract

DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Transfer Techniques*
Genetic Vectors*
Humans
Lentivirus / genetics*
MicroRNAs / antagonists & inhibitors*
MicroRNAs / genetics
Oligodeoxyribonucleotides / pharmacology*
Polymerase Chain Reaction / methods*
Signal Transduction
Transcription Factor AP-1 / metabolism

Substances

MicroRNAs
Oligodeoxyribonucleotides
Transcription Factor AP-1

Grants and funding

Funding provided by National Natural Science Foundation of China 81170047 and 81370151 to DG, http://www.nsfc.gov.cn/publish/portal1/tab131/; National Basic Research Program of China 973 Program 2012CB124701 to DG, http://www.most.gov.cn/eng/index.htm; Shenzhen Municipal Basic Research Program JC201006010725A to DG, http://www.szsti.gov.cn/; Shenzhen Overseas High-Level Talents Innovation Program YFZZ20111009 to DG, http://www.szsti.gov.cn/; Shenzhen High-tech Development Project CXZZ20140828163951592 to DG, http://www.szsti.gov.cn/; Transgenic project from the Ministry of Agriculture 2014ZX08009-051B to DG, http://english.agri.gov.cn/; Shenzhen Municipal Basic Research Program JCYJ20130329120507746 to KK, http://www.szsti.gov.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.