Silencing of the human SET gene in vitro with lentivirus-mediated RNA interference

Liu,Yanfeng; He,Pengcheng; Zhang,Mei; Shi,Lili; Zhu,Huachao; Wang,Yuan; Zhao,Jing

doi:10.3892/mmr.2013.1275

Silencing of the human SET gene in vitro with lentivirus-mediated RNA interference

Authors:
- Yanfeng Liu
- Pengcheng He
- Mei Zhang
- Lili Shi
- Huachao Zhu
- Yuan Wang
- Jing Zhao
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Affiliations: Department of Hematology, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, P.R. China
Published online on: January 15, 2013 https://doi.org/10.3892/mmr.2013.1275
Pages: 843-847

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Abstract

In our previous study, SET was identified as one of the differentially expressed proteins that was associated with tetra-arsenic tetra-sulfide (As4S4)-induced NB4-R1 [retinoic acid-resistant acute promyelocytic leukemia (APL) cell line] apoptosis. However, the mechanism through which SET regulates pathways during this process remains unclear. The aim of this study was to construct lentivirus-mediated short hairpin RNA (shRNA) against SET and investigate the effect of SET on As4S4-induced retinoic acid-resistant APL cell apoptosis. In the present study, 4 different oligonucleotides targeting the human SET gene were synthesized and cloned into the eukaryotic expression plasmid pGCSIL-GFP. The recombinant vectors were introduced into NB4-R1 cells. The silencing efficiency was measured by real-time quantitative PCR (RT-qPCR) and western blotting. Our results showed that the 4 recombinant RNA interference (RNAi) vectors were constructed successfully. Fluorescence microscopy demonstrated that infection efficiency ranged from 70 to 90%. Infection with the 4 different RNAi vectors significantly knocked down the expression of SET by 52.8, 69.1, 48.9 and 90.3% at the mRNA level, and 92.5, 96.3, 91.7 and 98.4% at the protein level, respectively. We attempt to clarify the mechanism of As4S4 treatment on retinoic acid-resistant APL.

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