Airway smooth muscle hyperproliferation is regulated by microRNA-221 in severe asthma

Abstract

Increased airway smooth muscle (ASM) mass is a feature of asthmatic airways, and could result from augmented proliferation. We determined whether proliferation and IL-6 release are abnormal in ASM cells (ASMCs) from patients with severe asthma, and whether these features could be mediated by microRNA-221 and microRNA-222, through modulation of the cyclin-dependent kinase inhibitors, p21(WAF1) and p27(kip1). ASMCs cultured from bronchial biopsies of healthy subjects and patients with nonsevere or severe asthma were studied. Proliferation was measured by the incorporation of bromodeoxyuridine and IL-6 by ELISA. FCS and transforming growth factor (TGF)-β caused greater proliferation and IL-6 release in patients with severe compared with nonsevere asthma and normal subjects. FCS + TGF-β inhibited p21(WAF1) and p27(kip1) expression, and increased microRNA-221 (miR-221) expression in ASMCs from individuals with severe asthma. miR-221, and not miR-222, mimics the increased proliferation and IL-6 release induced by FCS + TGF in healthy ASM, whereas in patients with severe asthma, the inhibition of miR-221, but not miR-222, inhibited proliferation and IL-6 release. miR-221 inhibition led to the increased expression of FCS + TGF-β-induced p21(WAF1) and p27(kip1). Dexamethasone suppressed proliferation in healthy subjects, but not in subjects with asthma. IL-6 was less suppressible by dexamethasone in patients with nonsevere and severe asthma, compared with healthy subjects. miR-221 did not influence the effects of dexamethasone. ASM from patients with severe asthma shows greater proliferation and IL-6 release than in patients with nonsevere asthma, but both groups show corticosteroid insensitivity. miR-221 regulates p21(WAF1) and p27(kip1) expression levels. Furthermore, miR-221 regulates the hyperproliferation and IL-6 release of ASMCs from patients with severe asthma, but does not regulate corticosteroid insensitivity.

Figure 1.

Effects of FCS and transforming growth factor–β (TGF-β) on airway smooth muscle (ASM) proliferation (A) and IL-6 release (B) from the ASM cells of healthy subjects, patients with nonsevere asthma, and patients with severe asthma at 8 days. Bars/points represent the means ± SEMs from nine ASM donors in each group. ^#P < 0.05. ⁺P < 0.05. ^##P < 0.01. ⁺⁺P < 0.01. ***P < 0.001. ⁺⁺⁺P < 0.001. ^###P < 0.001. Asterisks indicate comparison with no FCS control. Pound signs indicate comparison with healthy ASM cells. Plus signs indicate comparison with nonsevere ASM cells.

Figure 2.

Effects of FCS and TGF-β on the cyclin-dependent kinase inhibitors p21^WAF1 and p27^kip1 mRNA (B and C) and protein expression (A, D, and E) at 24 hours. (A) Examples of Western blots of protein expression from extracts of ASM cells exposed to control (No FCS), TGF-β, FCS, and [FCS+TGF-β] from one healthy subject, one subject with nonsevere asthma, and one subject with severe asthma for p21^WAF1, p27^Kip1, and β-actin. Each gel was probed with the three relevant antibodies separately, and each figure represents a composite gel of the four experimental conditions in each subject category. Protein quantification was normalized to control actin. (F and G) Correlated data of protein expression versus bromodeoxyuridine (BrdU) incorporation after stimulation with FCS and TGF-β. Bars represent the means ± SEMs of nine ASMC donors in each group. Open bars, healthy; shaded bars, nonsevere asthma; solid bars, severe asthma. Dotted lines represent 100%. *P < 0.05. **P < 0.01. ^##P < 0.01. ***P < 0.001.

Figure 3.

Effects of FCS and TGF-β on microRNA-221 (miR-221) and miR-222 expression between different subject groups at 24 hours. Mean miR-221 and miR-222 expressions, as measured by TaqMan RT-PCR, are shown in A and B, respectively. Bars represent the means ± SEMs of nine ASMC donors in each group. *P < 0.05. ^#P < 0.05. **P < 0.01.

Figure 4.

Effects of the overexpression of miR-221 and miR-222 in ASM cells from healthy subjects. DNA synthesis (A), IL-6 release (B), and p21^WAF1 (C) and p27^kip1 (D) mRNA expression were measured by BrdU ELISA, a DuoSet ELISA Assay (R&D Systems, Abingdon, UK), or TaqMan RT-PCR, respectively. DNA synthesis and IL-6 release were measured at 8 days, and mRNA expression was measured at 24 hours. Bars represent the means ± SEMs of nine ASMC healthy donors. “Neg” refers to no FCS. ^#P < 0.05. **P < 0.01. ^##P < 0.01. ***P < 0.001. ^###P < 0.001.

Figure 5.

Effects of inhibition of miR-221 and miR-222 in ASM cells from subjects with severe asthma. DNA synthesis (A), IL-6 release (B), and p21^WAF1 mRNA (C) and p27^kip1 mRNA (D) expression were subsequently measured by BrdU ELISA, a DuoSet ELISA Assay, or TaqMan RT-PCR, respectively. DNA synthesis and IL-6 release were measured at 8 days, and mRNA expression was measured at 24 hours. Bars represent the means ± SEMs of nine ASM donors with severe asthma. “Neg” refers to no FCS. ^#P < 0.05. **P < 0.01. ^##P < 0.01. ***P < 0.001. ^###P < 0.001.

Figure 6.

Effects of overexpression or inhibition of miR-221 and miR-222 on p21^WAF1 and p27^kip1 protein in ASM cells at 24 hours. Representative results from a subjects without asthma and from a subject with severe asthma are shown in A. Mean protein expression, quantified by densitometry, normalized against β-actin expression, and expressed as the percent change versus untreated control subjects, is shown in B–E. Bars represent the means ± SEMs of nine ASM donors in each group. “Neg” refers to no FCS. **P < 0.01. ^##P < 0.01. ^###P < 0.001.

Figure 7.

Effects of dexamethasone (Dex) on ASM proliferation, IL-6 release, p21^WAF1 and p27^kip1 mRNA, and miR-221 and miR-222 expression in ASM cells from healthy subjects, subjects with nonsevere asthma, and subjects with severe asthma. DNA synthesis (A) and IL-6 release (B) were measured by BrdU ELISA and DuoSet ELISA assays, respectively. C shows changes in p21^WAF1 and p27^kip1 mRNA expression, and D shows changes in miR-221 and miR-222 expression measured by TaqMan RT-PCR. DNA synthesis and IL-6 release were measured at 8 days, and mRNA and miRNA expression were measured at 24 hours. Bars/points represent the means ± SEMs from nine ASM donors in each group. *P < 0.05. ^#P < 0.05. **P < 0.01. ^##P < 0.01. ⁺⁺P < 0.01. ***P < 0.001. ^###P < 0.001. ⁺⁺⁺P < 0.001.