miR-221 suppresses ICAM-1 translation and regulates interferon-gamma-induced ICAM-1 expression in human cholangiocytes

Abstract

Aberrant cholangiocyte reactions in response to inflammatory stimuli are important pathogenic factors for the persistent biliary inflammation in patients with cholangiopathies. Overexpression of intercellular cell adhesion molecule-1 (ICAM-1) in cholangiocytes is a common pathological feature in inflammatory cholangiopathies and can promote cholangiocyte interactions with effector lymphocytes in the portal region. In this study, we tested the involvement of miRNA-mediated posttranscriptional regulation in IFN-gamma-induced ICAM-1 expression in cholangiocytes. Using both immortalized and nonimmortalized human cholangiocyte cell lines, we found that IFN-gamma activated ICAM-1 transcription and increased ICAM-1 protein expression. Inhibition of ICAM-1 transcription could only partially block IFN-gamma-induced ICAM-1 expression at the protein level. In silico target prediction analysis revealed complementarity of miR-221 to the 3'-untranslated region of ICAM-1 mRNA. Targeting of ICAM-1 3'-untranslated region by miR-221 resulted in translational repression in cholangiocytes but not ICAM-1 mRNA degradation. Functional inhibition of miR-221 with anti-miR-221 induced ICAM-1 protein expression. Moreover, IFN-gamma stimulation decreased miR-221 expression in cholangiocytes in a signal transducer and activator of transcription 1-dependent manner. Transfection of miR-221 precursor abolished IFN-gamma-stimulated ICAM-1 protein expression. In addition, miR-221-mediated expression of ICAM-1 on cholangiocytes showed a significant influence on the adherence of cocultured T cells. These findings indicate that both transcriptional and miRNA-mediated posttranscriptional mechanisms are involved in IFN-gamma-induced ICAM-1 expression in human cholangiocytes, suggesting an important role for miRNAs in the regulation of cholangiocyte inflammatory responses.

Fig. 1.

Interferon (IFN)-γ induces intercellular adhesion molecule (ICAM)-1 expression in cultured human cholangiocyte. A and B: dose-dependent expression of ICAM-1 at the message and the protein levels in H69 cells following IFN-γ stimulation. H69 cells were exposed to the culture medium with various doses of IFN-γ (0, 0.1, 1.0, 10, and 25 ng/ml) followed by real-time PCR (after incubation for 8 h) or Western blotting analysis for ICAM-1 (after incubation for 24 h). C and D: time-dependent expression of ICAM-1 expression in H69 cells induced by IFN-γ. Cells were exposed to IFN-γ (10 ng/ml) followed by real-time PCR (incubation for up to 24 h) or Western blot (incubation for up to 48 h). E and F: IFN-γ-induced expression of ICAM-1 in human intrahepatic biliary epithelial (HIBEpiC) cells. HIBEpiC cells were exposed to culture medium with or without IFN-γ (10 ng/ml) followed by real-time PCR (incubation for 8 h) or Western blot (incubation for 24 h). Bars represent the means ± SD from 3 independent experiments. *P < 0.05 t-test vs. the non-IFN-γ-stimulated control.

Fig. 2.

Posttranscriptional regulation is involved in IFN-γ-induced ICAM-1 protein expression in cholangiocytes. Expression of ICAM-1 at the message (A) and protein (B) levels in H69 cells following IFN-γ stimulation in the presence or absence of actinomycin D. H69 cells were exposed to culture medium with actinomycin D (10 μg/ml) for 90 min and then exposed to IFN-γ (10 ng/ml) following by real-time PCR for ICAM-1 (after incubation for 12 h) or Western blot (after incubation for 24 h). Data are representative of 3 independent experiments. *P < 0.05 t-test vs. non-IFN-γ-stimulated control.

Fig. 3.

miR-221 targets ICAM-1 3′-untranslated region (UTR) and causes posttranscriptional suppression. A: human ICAM-1 mRNA shows a potential binding site in the 3′UTR for miR-221. B: targeting of ICAM-1 3′UTR by miR-221 resulted in transcriptional suppression. The luciferase reporter constructs containing the potential binding site for miR-221 in ICAM-1 3′UTR or the mutant (Mut) sequence (TGTAGC to ACATCG) were generated. H69 cells were transiently cotransfected with the reporter construct and the miR-221 precursor or anti-miR-221 for 24 h. Luciferase activities were measured and normalized to the control (Ctrl) β-galactosidase (β-gal) level. A nonspecific precursor (precursor-Ctrl) and anti-miR (anti-miR-Ctrl) were used as the controls. Bars represent the means ± SD from 3 independent experiments. *P < 0.05 t-test vs. 3′UTR mutant; #P < 0.05 t-test vs. ICAM-1 3′UTR reporter construct.

Fig. 4.

Functional inhibition of miR-221 increases ICAM-1 protein expression but does not alter ICAM-1 mRNA level. A: anti-miR-221 increased ICAM-1 protein expression in cholangiocytes. H69 cells were treated with various doses of anti-miR-221 for 24 h followed by Western blot for ICAM-1. B: anti-miR-221 did not alter ICAM-1 expression at the message level in cholangiocytes. H69 cells were treated with various doses of anti-miR-221 for 24 h followed by real-time PCR for ICAM-1. Bars represent the means ± SD from 3 independent experiments. Anti-miR-Ctrl, nonspecific anti-miR control; *P < 0.05 t-test vs. non-anti-miR-221-treated cells.

Fig. 5.

IFN-γ decreases miR-221 expression in a signal transducer and activator of transcription (STAT)1-dependent manner. A and B: IFN-γ stimulation decreased miR-221 expression in cholangiocytes. H69 and HIBEpiC cells were exposed to IFN-γ (10 ng/ml) for 8 h or 12 h followed by Northern blot for miR-221 (in H69 cells in A) and real-time PCR (in H69 and HIBEpiC cells in B). RNU6B (U6) was used as the control. C: knockdown of STAT1 blocked IFN-γ-induced decrease of miR-221 expression. H69 cells were treated with either the STAT1 siRNA or the scrambled control siRNA for 48 h and then exposed to IFN-γ (10 ng/ml) for additional 8 h. Total RNA was isolated, and the expression of miR-221 was quantified by real-time PCR. Data are representative of 3 independent experiments. Bars represent the means ± SD from 3 independent experiments. *P < 0.05 t-test vs. non-IFN-γ-stimulated cells in B and C; #P < 0.05 t-test vs. the scrambled siRNA control in C.

Fig. 6.

IFN-γ induces ICAM-1 expression through downregulation of miR-221. A: IFN-γ increased the luciferase reporter translational activity in H69 cells transfected with the construct with ICAM-1 3′UTR encoding miR-221 binding site. Cells were transfected with the pMIR-REPORT luciferase construct containing the ICAM-1 3′UTR with the putative miR-221 binding site and then exposed to IFN-γ for 24 h. Luciferase activity in cells after exposure IFN-γ was then measured and normalized to β-gal. B: miR-221 precursor blocked IFN-γ-induced ICAM-1 protein expression. H69 cells were transfected with the miR-221 precursor or a control nonspecific precursor for 48 h and then exposed to IFN-γ (10 ng/ml) for 24 h followed by Western blot for ICAM-1. C: miR-221 precursor does not alter IFN-γ-induced ICAM-1 expression at the message level. H69 cells were transfected with the miR-221 precursor for 24 h and then exposed to IFN-γ (10 ng/ml) for 8 h followed by real-time PCR analysis for ICAM-1 mRNA. Data are representative of 3 independent experiments. Precursor-Ctrl, nonspecific precursor control. *P < 0.05 t-test vs. the empty vector control or 3′UTR mutant (in A) or non-IFN-γ-stimulated cells (in B and C); #P < 0.05 t-test vs. ICAM-1 3′UTR (in A) or non-miR-221 precursor-treated cells (in B).

Fig. 7.

Upregulation of ICAM-1 in cholangiocytes in response to IFN-γ affects adherence of cocultured T cells. A and B: functional manipulation of miR-221 influenced adherence of H69 by cocultured Jurkat cells in an ICAM-1-dependent manner. H69 cells were first treated with miR-221 precursor (A) or anti-miR-221 (B) for 72 h. After being washed, cells were incubated with the medium containing the IgG isotype control (Ctrl) or anti-ICAM-1 mAb for 1 h. Activated Jurkat cells were labeled with the calcein acetoxymethyl ester (calcein AM) and then incubated with H69 cells. Adherence of Jurkat cells was measured under the fluorescence microscope and presented as percentage of control. C: IFN-γ stimulation increased adherence of H69 cells by cocultured Jurkat cells. H69 cells were exposed to IFN-γ for 24 h followed by treatment with the anti-ICAM-1 mAb or IgG isotype for 1 h. H69 cells were treated with miR-221 precursor for 72 h before exposure to IFN-γ, and adherence of H69 cells by Jurkat cells was then determined. Data are representative of 3 independent experiments. Precursor-Ctrl, nonspecific precursor control. *P < 0.05 t-test vs. IgG isotype control (in A and B) or non-IFN-γ-stimulated cells (in C); #P < 0.05 t-test vs. precursor-Ctrl or IgG isotype control. D: effects of upregulation of ICAM-1 in H69 cells in response to IFN-γ on adherence of cocultured T cells as assessed by immunofluorescent microscopy. Calcein AM-labeled Jurkat cells following adherence to H69 cells were shown. Bars = 50 μm.