title: siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells.
abstract: Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14-siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14-siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14-siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.
authors.affiliation: College of Agriculture, Hainan Key Lab of Tropical Animal Reproduction & Breeding and Epidemic Disease Research, Hainan University, Haidian Island, Haikou, People's Republic of China.
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.forename: Hanwei
authors.initials: H
authors.lastname: Jiao
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Du
authors.forename: Ying
authors.initials: Y
authors.lastname: Cheng
authors.forename: Donglin
authors.initials: D
authors.lastname: Zhang
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Hao
authors.forename: Churiga
authors.initials: C
authors.lastname: Man
authors.forename: Fengyang
authors.initials: F
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D054426:Chemokine CXCL2 / Q000378:metabolism*; D020869:Gene Expression Profiling; D020868:Gene Silencing; D015850:Interleukin-6 / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide