Aberrant expression of interleukin-23-regulated miRNAs in T cells from patients with ankylosing spondylitis

Abstract

Background: Interleukin (IL)-23 can facilitate the differentiation of IL-17-producing helper T cells (Th17). The IL-23/IL-17 axis is known to play a key role in the immunopathogenesis of ankylosing spondylitis (AS). We hypothesized that the expression of microRNAs (miRNAs, miRs) would be regulated by IL-23 and that these miRNAs could participate in the immunopathogenesis of AS.

Methods: Expression profiles of human miRNAs in K562 cells, cultured in the presence or absence of IL-23 for 3 days, were analyzed by microarray. Potentially aberrantly expressed miRNAs were validated using T-cell samples from 24 patients with AS and 16 control subjects. Next-generation sequencing (NGS) was conducted to search for gene expression and biological functions regulated by specific miRNAs in the IL-23-mediated signaling pathway.

Results: Initial analysis revealed that the expression levels of 12 miRNAs were significantly higher, whereas those of 4 miRNAs were significantly lower, in K562 cells after coculture with IL-23 for 3 days. Among these IL-23-regulated miRNAs, the expression levels of miR-29b-1-5p, miR-4449, miR-211-3p, miR-1914-3p, and miR-7114-5p were found to be higher in AS T cells. The transfection of miR-29b-1-5p mimic suppressed IL-23-mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation in K562 cells. After NGS analysis and validation, we found that miR-29b-1-5p upregulated the expression of angiogenin, which was also upregulated in K562 cells after coculture with IL-23. Increased expression of miR-29b-1-5p or miR-211-3p could enhance interferon-γ expression.

Conclusions: Among the miRNAs regulated by IL-23, expression levels of five miRNAs were increased in T cells from patients with AS. The transfection of miR-29b-1-5p mimic could inhibit the IL-23-mediated STAT3 phosphorylation and might play a role in negative feedback control in the immunopathogenesis of AS.

Keywords: Angiogenin; Ankylosing spondylitis; IL-23; MicroRNAs; STAT3; T cells.

Fig. 1

Effect of interleukin (IL)-23 on signal transducer and activator of transcription 3 (STAT3) protein phosphorylation in K562 cells. a The phosphorylation ratio of STAT3 increased in K562 cells after coculture with IL-23 (20 ng/ml) for 3 days compared with those cocultured with medium (control) only. b A representative case

Fig. 2

Altered expression of interleukin (IL)-23-regulated microRNAs (miRNAs) in T cells from patients with ankylosing spondylitis (AS) and from healthy control subjects. a Expression profiles of miRNAs in K562 cells cocultured with or without IL-23 (20 ng/ml) for 3 days, evaluated using microarray analysis. Each scatter spot represents the mean raw signals of miRNA in three repeats of each treatment. b The expression levels of 12 miRNAs were significantly higher, whereas the expression levels of 4 miRNAs were significantly lower, in K562 cells after coculture with IL-23 for 3 days (P < 0.05). c Increased expression of miR-29b-1-5p, miR-4449, miR-211-3p, miR-1914-3p, and miR-7114-5p in AS T-cell miRNA, compared with normal T cells after validation

Fig. 3

Effect of miR-29b-1-5p on signal transducer and activator of transcription 3 (STAT3) protein phosphorylation in K562 cells. a Increased miR-29b-1-5p expression in K562 cells after transfection with miR-29b-1-5p mimic versus scramble oligonucleotides. b The phosphorylation ratio of STAT3 decreased in K562 cells after transfection with miR-29b-1-5p mimic compared with those transfected with scramble oligonucleotides after culturing with medium for 48 h. c A representative case

Fig. 4

Identification of miR-29b-1-5p-regulated genes. a Expression profiles of messenger RNAs (mRNAs) in K562 cells transfected with miR-29b-1-5p mimic or scramble oligonucleotides then cultured with medium for 48 h were evaluated using RNA-Seq transcriptome analysis. Each scatter spot represents the mean raw signals of microRNA (miRNA) in three repeats of each treatment. b The RNA expression levels of 16 genes were significantly higher, whereas the RNA expression levels of 10 genes were significantly lower, in K562 cells after being transfected with miR-29b-1-5p mimic for 48 h. c Four genes potentially involved in the inflammatory responses were selected for further analysis. The mRNA expression levels of ANG were significantly elevated in K562 cells after coculture with interleukin (IL)-23 (20 ng/ml) for 3 days (Fig. 3c)

Fig. 5

Protein expression of angiogenin was regulated by miR-29b-1-5p and interleukin (IL)-23. a The protein expression of angiogenin was elevated in K562 cells after being transfected with miR-29b-1-5p mimic for 48 h compared with those transfected with scramble oligonucleotides. b A representative case. c The protein expression of angiogenin was also increased in K562 cells after coculture with IL-23 for 3 days compared with those cultured with medium only. d A representative case

Fig. 6

Effect of miR-29b-1-5p on proinflammatory cytokine expression. a The messenger RNA (mRNA) expression levels of interferon-γ (IFN-γ), but not interleukin (IL)-17A or tumor necrosis factor-α (TNF-α), were increased in K562 cells 24 h after transfection of miR-29b-1-5p mimic compared with those transfected with scrambled oligonucleotides. b The mRNA expression levels of IFN-γ, but not IL-17A or TNF-α, were increased in K562 cells after coculture with IL-23 (20 ng/ml) compared with those cultured with medium alone. c The K562 cells transfected with miR-29b-1-5p mimic or scrambled oligonucleotides were cultured with IL-23 (20 ng/ml) for 24 h, and there was no difference in the mRNA expression levels of IFN-γ, IL-17A, or TNF-α between these two groups

Fig. 7

Effect of miR-211-3p in proinflammatory cytokine expression. a The expression levels of miR-211-3p dramatically increased after transfection with miR-211-3p mimic compared with those transfected with scrambled oligonucleotides (control). b There were no differences in the mRNA expression levels of interferon-γ (IFN-γ), interleukin (IL)-17A, or tumor necrosis factor-α (TNF-α) in K562 cells 24 h after transfection of miR-211-3p mimic compared with those transfected with scrambled oligonucleotides. c The K562 cells transfected with miR-211-3p mimic or scrambled oligonucleotides were cultured with IL-23 (20 ng/ml) for 24 h. The mRNA expression levels of IFN-γ, but not IL-17A or TNF-α, were increased in those transfected with miR-211-3p mimic compared with the control