Lipopolysaccharide‐induced miR‐1224 negatively regulates tumour necrosis factor‐α gene expression by modulating Sp1

Abstract

Summary 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.

Lipopolysaccharide Decreases Single Immunoglobulin Interleukin-1 Receptor-related Molecule (SIGIRR) Expression by Suppressing Specificity Protein 1 (Sp1) via the Toll-like Receptor 4 (TLR4)-p38 Pathway in Monocytes and Neutrophils

“…4, C and D, caffeic acid phenethyl ester (CAPE)). There is another conceptually novel report identifying some microRNAs as negative regulators for LPS-dependent target genes (28,29). Among these microRNAs, miR1224 is likely a key inhibitor of Sp1 mRNA expression (29).…”

Complexity of microRNA function and the role of isomiRs in lipid homeostasis

“…For example, a human miRtron, miR-1224, was found to be induced by lipopolysaccahride (LPS) and to directly target and regulate Sp1 transcription factor activity, which controls the expression of many lipid-related genes ( 77,104 ) including the LDL receptor (105)(106)(107). Another recent study found that miR-451, which bypasses DICER processing, is signifi cantly reduced in the livers of rats on high-fat diets ( 108 ), supporting an earlier study that found hepatic miR-451 to be downregulated in humans with nonalcoholic steatohepatitis (NASH) ( 109 ).…”

The expression of microRNA-155 and 127 in the neonatal SD rats with acute lung injury induced by lipopolysaccharide

“…Protein concentrations were determined using a Bradford reagent (BioRad Laboratories, Inc.). Western blotting was performed as previously described [13], and the following primary antibodies and dilutions used were: anti-TNF-α (1:1,000 dilution; Beyotime Institute of Biotechnology, China), anti-IL-6 (1:1,000 dilution; Beyotime Institute of Biotechnology, China) and anti-β-actin (1:1,000 dilution; Beyotime Institute of Biotechnology, China).…”