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

doi:10.1194/jlr.M400328-JLR200

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

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

Diabetes, Beckman Research Institute of City of Hope, Duarte, CA 91010

Corresponding Author: RNatarajan{at}coh.org

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 monocyte/macrophages, endothelial cells and vascular smooth muscle cells (VSMC). Our objective was therefore to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance macrophages and VSMC. We utilized 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 (EGFP). We then cloned these U6 promoter+siRNA PCR products into plasmid vectors (shRNAs) to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMC. 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 reduced cellular adhesion and fibronectin expression in VMSC. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, Vascular cell adhesion molecule-1 and interleukin-6 in VSMC. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMC and its relationship to oxidant stress and inflammation.

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