title: A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.
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.
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.|Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Qiu
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Jiasheng
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Luo
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Nian
authors.initials: N
authors.lastname: Liu
authors.affiliation: Department of Physiology, Shenzhen University Health Science Center, Shenzhen, Guangdong, 518000, China.
authors.forename: Kang
authors.initials: K
authors.lastname: Kang
authors.affiliation: Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Junle
authors.initials: J
authors.lastname: Qu
authors.affiliation: Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Wenda
authors.initials: W
authors.lastname: Peng
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China.
authors.forename: Deming
authors.initials: D
authors.lastname: Gou
mesh_terms: D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D009838:Oligodeoxyribonucleotides; D018808:Transcription Factor AP-1