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.

Figure 1

Up-regulated microRNAs (miRNAs) obtained for microarray scanning were confirmed by real-time PCR. Total RNA extracted from spleen of mouse injected with saline or lipopolysaccharide (LPS) was analysed by using stem-loop real-time PCR. Relative expression levels of miRNAs were normalized to the level of U6 in each sample, and data are shown with miRNAs expression in control set as one.

Figure 2

MicroRNAs (miRNAs) were up-regulated in RAW264.7 cells upon stimulation with lipopolysaccharide (LPS). (a) Tumour necrosis factor-α (TNF-α) protein secretion was dramatically increased and reached a peak at 12 hr after addition of LPS. RAW264.7 cells were cultured in medium and treated with LPS at a final concentration of 1 μg/ml. TNF-α protein levels in supernatant were measured by ELISA. (b) The concentration ratio of TNF-α protein secreted from LPS-treated RAW264.7 cells to untreated RAW264.7 cells. (c, d) Relative expression levels of TNF and TLR4 mRNAs in response to LPS. (e–g) Relative expression levels of miR-146b, miR-155 and miR-1224 in RAW264.7 cells treated with LPS. (h) Semi-quantitative PCR for miRNAs and mRNAs.

Figure 3

Quantitative real-time PCR analysis of microRNA (miRNA) tissue expression pattern. Stem-loop reverse transcription-PCR primers sequences to mature miRNAs and U6 are shown in Table 1. The results were normalized against U6, and the relative expression is shown with miRNA expression in the heart set as one.

Figure 4

miR-1224 decreases tumour necrosis factor-α (TNF-α) promoter activity. Luciferase reporters driven by TNF-α core promoter were co-transfected with pRL-TK and miRNA-1224 mimics into RAW264.7 cells. Firefly and Renilla luciferase activities were quantified using the dual-luciferase reporter assay system. The results are shown as the mean ± SD of three individual samples and the graphs are representative of three independent experiments.

Figure 5

Effect of microRNA (miRNA) mimics and miRNA inhibitors on nuclear factor-κB (NF-κB) activity. Luciferase reporters driven by NF-κB response elements were co-transfected with pRL-TK and either mimics or inhibitors into RAW264.7 cells. Firefly and Renilla luciferase activities were quantified using the dual-luciferase reporter assay system. (a, b) Results demonstrate that miR-146b decreased the basal activity of NF-κB; miR-155 and miR-1224 increased the basal activity of NF-κB in RAW264.7 cells. (c) miR-1224 decreased NF-κB (p65) protein level in nucleus. Nucleoproteins were extracted from RAW264.7 cells transfected with miR-1224 mimics and miR-1224 inhibitor, respectively, and NF-κB (p65) protein levels were evaluated by Western blot analysis.

Figure 6

MicroRNA (miRNA) -gene regulatory network. Prediction of miRNA putative target genes was performed using TargetScan software in GeneSpring. Predicted target genes that associated with lipopolysaccharide (LPS) signalling target and miRNAs were integrated into a network map.

Figure 7

Micro(mi)R-1224 silences Sp1 by repressing its translation. (a) Alignment of potential miR-1224 binding sites in 3′ untranslated region (UTR) of Sp1 mRNA of different species. (b) The target sequence for miR-1224 at Sp1 3′ UTR. A 59-bp fragment bearing miR-1224 target binding site was chemically synthesized and inserted into psicheck-2 vector. (c) Mutant of the target sequence for miR-1224 at Sp1 3′ UTR. * indicates mutation sites. (d) Verification of interaction between miR-1224 and the 3′ UTR of Sp1 in HEK293 cells, determined by luciferase reporter activity. Ctl, control without transfected with miR-1224 mimics; Mimics Ctl, mimics negative control. Error bars indicate SD. *P < 0·05 versus mimics Ctl. (e) Western blot analysis of Sp1 protein expression in RAW264.7 cells transfected with miR-1224 mimics or control. Inhibitor-1224, single stranded nucleic acids designed to specifically bind to and functionally inhibit miR-1224. (f) Inhibitory role of miR-1224 on tumour necrosis factor-α (TNF-α).can be rescued by Sp1. Vector, pcDNA3.1 empty vector. Error bars indicates SD 5% level of significance of difference marked with lowercase letters of a, b, c. Significant difference between the two averages indicated by different letters, and the average difference between the two was not significant where letters are the same.

Figure 8

Conformation of inhibitory effect of micro(mi)R-1224 on tumour necrosis factor-α (TNF-α) promoter by examining Sp1 binding in vivo. DNA-Sp1 complexes from RAW264.7 cells transfected with mimics control or miR-1224 were immunoprecipitated with anti-Sp1 antibody. DNA purified from each sample was used for real-time PCR and semi-RT-PCR. (a) Real-time PCR analysis of Sp1 binding to in TNF-α promoter region. 1: Mimics control transfection input. 2: Mimics control transfection. 3: miR-1224 transfection input. 4: miR-1224 transfection. (b) Semi-RT-PCR analysis of Sp1 binding to in TNF-α promoter region. Lane 1: one-tenth of total input for sample transfected with mimics control; lane 2: one-tenth of total input for sample transfected with miR-1224 mimics; lanes 3 and 5: sample transfected with mimics-Ctl; lanes 4 and 6: sample transfected with miR-1224; lane 7: no template control; F1/R1: TNF-α promoter specific primers; F2/R2: negative primer, flanking far away from Sp1 binding site.