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

Yanfeng Liu1, Pengcheng He, Mei Zhang

  • 1Department of Hematology, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, PR China.

Molecular Medicine Reports
|January 23, 2013
PubMed

Insights

Researchers investigated the role of SET protein in tetra-arsenic tetra-sulfide (As4S4) induced apoptosis in retinoic acid-resistant acute promyelocytic leukemia (APL) cells. Silencing SET significantly enhanced As4S4-induced apoptosis, clarifying its mechanism in APL treatment.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Cell Biology

Background:

  • SET protein is differentially expressed in tetra-arsenic tetra-sulfide (As4S4)-induced apoptosis of retinoic acid-resistant acute promyelocytic leukemia (APL) NB4-R1 cells.
  • The precise mechanism by which SET influences As4S4-induced apoptosis in APL remains to be elucidated.

Purpose of the Study:

  • To construct lentivirus-mediated short hairpin RNA (shRNA) targeting the SET gene.
  • To investigate the effect of SET knockdown on As4S4-induced apoptosis in retinoic acid-resistant APL cells.

Main Methods:

  • Synthesis and cloning of four distinct oligonucleotides targeting the human SET gene into a eukaryotic expression plasmid.
  • Introduction of recombinant vectors into NB4-R1 cells.
  • Measurement of SET silencing efficiency using real-time quantitative PCR (RT-qPCR) and western blotting.

Main Results:

  • Successful construction of four recombinant RNA interference (RNAi) vectors targeting SET.
  • High infection efficiency (70-90%) demonstrated by fluorescence microscopy.
  • Significant knockdown of SET expression at both mRNA (48.9-90.3%) and protein (91.7-98.4%) levels.

Conclusions:

  • Lentivirus-mediated shRNA is effective in silencing SET expression in NB4-R1 cells.
  • SET plays a crucial role in regulating As4S4-induced apoptosis in retinoic acid-resistant APL.
  • This study provides insights into the mechanism of As4S4 treatment in APL.

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