Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs.
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.
Li
Shu-Lian
SL
Gonda Diabetes Research Center, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.
Dwarakanath
Roopashree S
RS
Cai
Qiangjun
Q
Lanting
Linda
L
Natarajan
Rama
R
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000494:pharmacology; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation; D008077:Lipoproteins, LDL / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
C475064:12-15-lipoxygenase; D018932:Chemokine CCL2; D018925:Chemokines; D017931:DNA Primers; D018076:DNA, Complementary; D005353:Fibronectins; D008077:Lipoproteins, LDL; D016877:Oxidants; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C067883:dihydroethidium; D013481:Superoxides; D049452:Green Fluorescent Proteins; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D004996:Ethidium