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

• Published in: Journal of lipid research 2005-02, Vol.46 (2), p.220-229
• Main Authors: Li, Shu-Lian, Dwarakanath, Roopashree S., Cai, Qiangjun, Lanting, Linda, Natarajan, Rama
• Format: Article
• Language: English
• Subjects: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid - pharmacology; Animals; Arachidonate 12-Lipoxygenase - genetics; Arachidonate 12-Lipoxygenase - metabolism; Arachidonate 15-Lipoxygenase - genetics; Arachidonate 15-Lipoxygenase - metabolism; arachidonic acid; atherosclerosis; Base Sequence; Blotting, Western; Cell Adhesion; Cell Line; Cell Movement; Cells, Cultured; Chemokine CCL2 - metabolism; Chemokines - metabolism; DNA Primers - chemistry; DNA, Complementary - metabolism; Down-Regulation; Endothelium, Vascular - metabolism; Ethidium - analogs & derivatives; Ethidium - pharmacology; Fibronectins - chemistry; Fibronectins - metabolism; Gene Silencing; Green Fluorescent Proteins - metabolism; Humans; Immunoblotting; Inflammation; inflammatory genes; Lipoproteins, LDL - metabolism; macrophages; Macrophages - metabolism; Mice; Microscopy, Fluorescence; Molecular Sequence Data; monocyte chemoattractant protein-1; monocytes; Monocytes - metabolism; Myocytes, Smooth Muscle - cytology; Oxidants - metabolism; Oxidative Stress; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Binding; Rats; Recombinant Fusion Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA interference; RNA, Messenger - metabolism; RNA, Small Interfering - metabolism; Superoxides - metabolism; Transfection; vascular smooth muscle cells
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.M400328-JLR200

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.