Role of non-coding RNAs in maintaining primary airway smooth muscle cells

Abstract

Background: The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases.

Objective: We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS).

Methods: mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR.

Results: A small number of miRNAs (including miR-150, -371-5p, -718, -940, -1181, -1207-5p, -1915, and -3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA 'sponges' for 4 miRNAs (miR-150, -371-5p, -940, -1207-5p).

Conclusion: This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD).

Figure 1

Effect of dexamethasone and FCS upon primary ASM cell proliferation, IL-6 and CXCL8 release. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. DNA synthesis (A), cell viability (B), IL-6 (C) and CXCL8 (D) release were measured by BrdU ELISA, DuoSet ELISA or MTT assay, respectively. Bars represent mean ± SEM from 9 primary ASM cells. ^***/###p < 0.001.

Figure 2

Effect of dexamethasone and FCS upon mRNA expression in primary ASM cells. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. To validate the array data, the expression of 5 mRNAs were confirmed by TaqMan RT-PCR. Bars represent mean ± SEM from 9 primary ASM cells. ⁺⁺⁺p < 0.001 vs. 18S; ^*/#p < 0.05 vs. baseline; ^**/##p < 0.01 vs. baseline; ^###p < 0.001 vs. baseline.

Figure 3

Effect of dexamethasone and FCS upon miRNA expression in primary ASM cells. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. To validate the array data, the expression of 6 miRNAs were confirmed by TaqMan RT-PCR. Bars represent mean ± SEM from 9 primary ASM cells. ^*/#p < 0.05 vs. baseline.

Figure 4

Effect of dexamethasone and FCS upon the predicted mRNA targets of miRNAs decreased in expression in primary ASM cells. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. 9 mRNAs were changed in expression following treatment. To validate the array data, the expression of these mRNAs were confirmed by TaqMan RT-PCR. Bars represent mean ± SEM from 9 primary ASM cells. ^*/#p < 0.05 vs. baseline; ^**/##p < 0.01 vs. baseline; ^###p < 0.001 vs. baseline.

Figure 5

Effect of dexamethasone and FCS upon lncRNA expression in primary ASM cells. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. To validate the array data, the expression of LINC00882, LINC00883, c-MYC and PVT1 were confirmed by TaqMan RT-PCR. Bars represent mean ± SEM from 9 primary ASM cells. ^*/#p < 0.05.

Figure 6

Proposed ncRNA interactions in primary airway smooth muscle cells following treatment with dexamethasone before stimulation with FCS. ASM cells were incubated with dexamethasone (10⁻⁷ M) for 1 h before being stimulated with FCS (2.5%) for 24 h. The lincRNAs RP11-46A10.4, LINC00883, BCYRN1 and LINC00882 act as ‘sponges’ for the miRNAs −1207, −150, −940 and −371. This, in turn, allows for the mRNA targets of these miRNAs to be expressed.