MicroRNA-155 acts as an anti-inflammatory factor in orbital fibroblasts from Graves' orbitopathy by repressing interleukin-2-inducible T-cell kinase

Abstract

To investigate the role of microRNA (miR)-155 in inflammation in an in-vitro model of Graves' orbitopathy (GO). The expression levels of miR-155 were compared between GO and non-GO orbital tissues. The effects of inflammatory stimulation of interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) on miR-155 expression on GO and non-GO orbital fibroblasts (OFs) were investigated. The effects of miR-155 mimics and inhibitors of inflammatory proteins and IL-2-inducible T-cell kinase (ITK) expression were examined, along with those related to the knockdown of ITK with siITK transfection on inflammatory proteins. We also examined how ITK inhibitors affect miR-155 expression in GO and non-GO OFs. The expression levels of miR-155 were higher in GO orbital tissues than in non-GO tissue. The overexpression of miR-155 was induced by IL-1β and TNF-α in OFs from GO and non-GO patients. IL-1β-induced IL-6 (ICAM1) protein production was significantly reduced (increased) by miR-155 mimics and inhibitors. The mRNA and protein levels of ITK were downregulated by overexpressed miR-155 via miR-155 mimics. Knockdown of ITK via siITK transfection induced a decrease in the expression levels of ITK, IL-17, IL-6, IL-1β, and TNF-α protein. The expression of miR-155 was significantly downregulated by treatment with ITK inhibitors and Bruton's tyrosine kinase (BTK)/ITK dual inhibitors in a time-dependent manner. Our results indicated a potential relationship between miR-155 and ITK in the context of GO OFs. The overexpression of miR-155 repressed ITK expression and relieved inflammation. Thus, miR-155 appears to have anti-inflammatory effects in GO OFs. This discovery provides a new concept for developing GO treatment therapeutics.

Fig 1. Expression of microRNA (miR)-155 in the orbital tissue of patients with Graves’ orbitopathy (GO, n = 12) and non-GO patients (n = 10).

(*P < 0.05, comparison between GO and non-GO in orbital adipose tissues).

Fig 2. Effects of interleukin (IL)-1β and tumour necrosis factor-alpha (TNF-α) on miR-155 expression in GO (n = 5) and non-GO orbital fibroblasts (n = 5) (OFs).

Note that IL-1β and TNF-α tend to induce an increase in miR-155 expression. (A, B) Time-dependent effects of IL-1β and TNF-α, (C, D) Dose dependent effects of IL-1β and TNF-α. Differences between treated and untreated cells are indicated (*P < 0.05, versus time and concentration matched controls).

Fig 3. Effects of microRNA-155 mimics and inhibitors on interleukin (IL)-1β induced IL-6, cyclooxygenase (COX)-2, and intercellular adhesion molecule (ICAM)-1 protein and mRNA expression in Graves’ orbitopathy (GO) (n = 4) orbital fibroblasts (OFs).

(A, B) IL-1β-induced IL-6 and ICAM1 protein production was reduced by miR-155 mimics, and increased by miR-155 inhibitors, respectively. (*P < 0.05) (C) IL-1β-induced IL-6 and ICAM1 mRNA expression were reduced by miR-155 mimics, and IL-1β-induced COX2 and ICAM1 mRNA expression was increased by miR-155 inhibitors. (*P < 0.05) These experiments were performed at least three times using OFs from at least three GO patients. The results are expressed as the mean ± standard deviation of at least three individual samples, and the graphs are representative of three independent experiments.

Fig 4

(A) The predicted binding site of microRNA (miR)-155 in the 3’-UTR of interleukin-2-inducible T-cell kinase (ITK) is indicated. (B, C) Effects of miR-155 mimics and inhibitors on ITK mRNA and protein expression in Graves’ orbitopathy (n = 3) orbital fibroblasts. (**P < 0.01) The results are expressed as the mean ± standard deviation of at least three individual samples, and the graphs are representative of three independent experiments.

Fig 5. Effects of inhibition of interleukin (IL)-2- inducible T-cell kinase (ITK) on the expression of inflammatory cytokines in Graves’ orbitopathy (GO) (n = 4) orbital fibroblasts (OFs).

(A, B) Effects of siITK on the expression levels of ITK, IL-17, IL-6, IL-1β, and tumour necrosis factor-alpha protein, revealed using Western blotting. (*P < 0.05) The results are expressed as the mean ± standard deviation of at least three individual samples, and the graphs are representative of three independent experiments. (C) Quantitative real-time polymerase chain reaction, showed that the mRNA expression of IL-17 was suppressed by siITK treatment. (**P < 0.01) These experiments were performed at least three times using OFs from at least three GO individuals.

Fig 6. Effects of inhibitors of extracellular signal-regulated kinase, nuclear factor kappa-light-chain-enhancer of activated B cell, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase/Akt, P38 mitogen activated protein kinase, and mammalian target of rapamycin on microRNA-155 expression in Graves’ orbitopathy (n = 3) orbital fibroblasts.

(*P < 0.05, **P < 0.01).

Fig 7. Effect of interleukin-2-inducible T-cell kinase (ITK) inhibitors and Bruton’s tyrosine kinase (BTK)/ITK dual inhibitors on the viability and microRNA-155 expression of orbital fibroblasts in Graves’ orbitopathy (GO) (n = 3) and non-GO (n = 3).

(A) Based on the MTT results, exposure of cells to ≤ 1 μM of ITK inhibitors and BTK/ITK dual inhibitors for 24 hours did not reduce cell viability to levels below 80% in GO OFs or 90% in non-GO OFs. (B) The expression of miR-155 was significantly downregulated by treatment with ITK inhibitors and BTK/ITK dual inhibitors, in a time-dependent manner in both GO and non-GO OFs. Differences between treated and untreated cells are indicated (in contrast to cells no treated with ITK, BTK/ITK inhibitors, *P < 0.05).