Transcriptional profiling identifies the long noncoding RNA plasmacytoma variant translocation (PVT1) as a novel regulator of the asthmatic phenotype in human airway smooth muscle

Abstract

Background: The mechanism underlying nonsevere and severe asthma remains unclear, although it is commonly associated with increased airway smooth muscle (ASM) mass. Long noncoding RNAs (lncRNAs) are known to be important in regulating healthy primary airway smooth muscle cells (ASMCs), whereas changed expression has been observed in CD8 T cells from patients with severe asthma.

Methods: Primary ASMCs were isolated from healthy subjects (n = 9) and patients classified as having nonsevere (n = 9) or severe (n = 9) asthma. ASMCs were exposed to dexamethasone and FCS. mRNA and lncRNA expression was measured by using a microarray and quantitative real-time PCR. Bioinformatic analysis was used to examine relevant biological pathways. Finally, the lncRNA plasmacytoma variant translocation 1 (PVT1) was inhibited by transfection of primary ASMCs with small interfering RNAs, and the effect on ASMC phenotype was examined.

Results: The mRNA expression profile was significantly different between patient groups after exposure to dexamethasone and FCS, and these were associated with biological pathways that might be relevant to the pathogenesis of asthma, including cellular proliferation and pathways associated with glucocorticoid activity. We also observed a significant change in lncRNA expression, yet the expression of only one lncRNA (PVT1) is decreased in patients with corticosteroid-sensitive nonsevere asthma and increased in patients with corticosteroid-insensitive severe asthma. Subsequent targeting studies demonstrated the importance of this lncRNA in controlling both proliferation and IL-6 release in ASMCs from patients with severe asthma.

Conclusions: lncRNAs are associated with the aberrant phenotype observed in ASMCs from asthmatic patients. Targeting PVT1 might be effective in reducing airway remodeling in asthmatic patients.

Keywords: Asthma; IL-6; PVT1; airway smooth muscle; long noncoding RNA; proliferation; transcriptome.

Fig E1

Fig E2

Fig 1

Venn diagrams showing pathway analysis of differentially expressed mRNAs in ASM from asthmatic patients. A-C, Pathway analysis showing intergroup comparisons at baseline (Fig 1, A), after stimulation with FCS (2.5%; Fig 1, B), and after stimulation with FCS (2.5%) plus TGF-β (1 ng/mL; Fig 1, C). Data represent 9 members of each patient type. D, Expression of 9 mRNAs was confirmed by using TaqMan RT-PCR to validate the array data. Bars represent means ± SEMs from 9 primary ASMC donors. *P < .05, **P < .01, and ***P < .001.

Fig 2

Effect of dexamethasone and FCS plus TGF-β on PVT1 lncRNA expression in ASMCs from asthmatic patients. A, FCS plus TGF-β–induced PVT1 lncRNA expression was measured by using quantitative RT-PCR over 1, 3, 6, and 24 hours. B, Dexamethasone (Dex) and FCS plus TGF-β–induced PVT1 expression was measured by using quantitative RT-PCR at 24 hours. Points/bars represent means ± SEMs of 9 ASMC donors. **P < .01 and ***P < .001. ##P < .01.

Fig 3

Effect of targeting PVT1 with siRNAs on IL-6 release and BrdU incorporation in ASMCs from healthy subjects and patients with severe asthma. A and B,PVT1 lncRNA expression was measured by using RT-PCR after ASM from healthy subjects and patients with severe asthma was transfected with siRNA (300 nmol/L), which was designed to target PVT1. C-F, BrdU incorporation (Fig 3, C and D) and IL-6 release (Fig 3, E and F) were measured by using the BrdU ELISA and DuoSet ELISA, respectively, at 8 days. Bars/points represent means ± SEMs of 9 ASMC donors. */#P < .05, ##/++P < .01, and ***/###P < .001. Dex, Dexamethasone.

Fig 4

Effect of targeting PVT1 with siRNAs on IL6 and c-MYC mRNA in ASMCs from healthy subjects and patients with severe asthma. A and B,IL6 and c-MYC mRNA expression was measured by using RT-PCR after exposure to dexamethasone (Dex; 10⁻⁷ mol/L) and stimulation with FCS (2.5%) plus TGF-β (1 ng/mL) for 24 hours. C-F, ASMCs from healthy subjects and patients with severe asthma were transfected with siRNAs designed to target PVT1. Also, the expression of IL6 (Fig 4, C and E) and c-MYC (Fig 4, D and F) mRNA was measured by using RT-PCR. Bars represent means ± SEMs of 9 ASMC donors. #P < .05, **/##/++P < .01, and ***/###/+++P < .001.

Fig 5

A and B, Potential mechanisms for PVT1 contribution to ASMC proliferation and IL-6 release in asthmatic patients. Dex, Dexamethasone; FCS, fetal calf serum; TGF-β, transforming growth factor-beta.