title: Lipopolysaccharide-induced miR-1224 negatively regulates tumour necrosis factor-α gene expression by modulating Sp1.
abstract: The innate immune response provides the initial defence mechanism against infection by other organisms. However, an excessive immune response will cause damage to host tissues. In an attempt to identify microRNAs (miRNAs) that regulate the innate immune response in inflammation and homeostasis, we examined the differential expression of miRNAs using microarray analysis in the spleens of mice injected intraperitoneally with lipopolysaccharide (LPS) and saline, respectively. Following challenge, we observed 19 miRNAs up-regulated (1.5-fold) in response to LPS. Among these miRNAs, miR-1224, whose expression level increased 5.7-fold 6 hr after LPS injection and 2.3-fold after 24 hr, was selected for further study. Tissue expression patterns showed that mouse miR-1224 is highly expressed in mouse spleen, kidney and lung. Transfection of miR-1224 mimics resulted in a decrease in basal tumour necrosis factor-α (TNF-α) promoter reporter gene activity and a down-regulation of LPS-induced TNF-α mRNA in RAW264.7 cells. With public databases of miRNA target prediction, miR-1224 was shown to bind to the 3' untranslated region (UTR) of Sp1 mRNA, whose coding product controls TNF-α expression at the transcriptional level. Furthermore, we found that in HEK-293 cells, the activity of the luciferase reporter bearing Sp1 mRNA 3' UTR was down-regulated significantly when transfected with miR-1224 mimics. After transfection of miR-1224 in RAW264.7 cells, nucleus Sp1 protein level decreased, and when endogenous miR-1224 was blocked, the decrease was abolished. Therefore, we initially speculated that miR-1224 was a negative regulator of TNF-α in an Sp1-dependent manner, which was confirmed in vivo by chromatin immunoprecipitation assay, and might be involved in regulating the LPS-mediated inflammatory responses.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Science, Sun Yat-Sen University, Guangzhou, Guangdong, China. niuyuna@genomics.org.cn
authors.forename: Yuna
authors.initials: Y
authors.lastname: Niu
authors.forename: Delin
authors.initials: D
authors.lastname: Mo
authors.forename: Limei
authors.initials: L
authors.lastname: Qin
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Anning
authors.initials: A
authors.lastname: Li
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhao
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Wang
authors.forename: Shuqi
authors.initials: S
authors.lastname: Xiao
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Xie
authors.forename: Zuyong
authors.initials: Z
authors.lastname: He
authors.forename: Peiqing
authors.initials: P
authors.lastname: Cong
authors.forename: Yaosheng
authors.initials: Y
authors.lastname: Chen
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics / Q000276:immunology; D000818:Animals; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000276:immunology*; D007113:Immunity, Innate / Q000235:genetics* / Q000276:immunology; D007136:Immunoglobulins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D046228:Microarray Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D020413:3' Untranslated Regions; D007136:Immunoglobulins; D008070:Lipopolysaccharides; C558413:MIRN1224 microRNA, mouse; D035683:MicroRNAs; C090698:SP1 antigen; D014409:Tumor Necrosis Factor-alpha