Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs

Shu Lian Li, Roopashree S. Dwarakanath, Qiangjun Cai, Linda Lanting, Rama Natarajan

Research output: Contribution to journalArticle

31 Scopus citations

Abstract

The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.

Original languageEnglish (US)
Pages (from-to)220-229
Number of pages10
JournalJournal of Lipid Research
Volume46
Issue number2
DOIs
StatePublished - Feb 1 2005

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Keywords

  • Arachidonic acid
  • Atherosclerosis
  • Inflammatory genes
  • Macrophages
  • Monocyte chemoattractant protein-1
  • Monocytes
  • RNA interference
  • Vascular smooth muscle cells

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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