High Stretch Modulates cAMP/ATP Level in Association with Purine Metabolism via miRNA-mRNA Interactions in Cultured Human Airway Smooth Muscle Cells

Abstract

High stretch (>10% strain) of airway smooth muscle cells (ASMCs) due to mechanical ventilation (MV) is postulated to contribute to ventilator-induced lung injury (VILI), but the underlying mechanisms remain largely unknown. We hypothesized that ASMCs may respond to high stretch via regulatory miRNA-mRNA interactions, and thus we aimed to identify high stretch-responsive cellular events and related regulating miRNA-mRNA interactions in cultured human ASMCs with/without high stretch. RNA-Seq analysis of whole genome-wide miRNAs revealed 12 miRNAs differentially expressed (DE) in response to high stretch (7 up and 5 down, fold change >2), which target 283 DE-mRNAs as identified by a parallel mRNA sequencing and bioinformatics analysis. The KEGG and GO analysis further indicated that purine metabolism was the first enriched event in the cells during high stretch, which was linked to miR-370-5p-PDE4D/AK7. Since PDE4D/AK7 have been previously linked to cAMP/ATP metabolism in lung diseases and now to miR-370-5p in ASMCs, we thus evaluated the effect of high stretch on the cAMP/ATP level inside ASMCs. The results demonstrated that high stretch modulated the cAMP/ATP levels inside ASMCs, which could be largely abolished by miR-370-5p mimics. Together, these findings indicate that miR-370-5p-PDE4D/AK7 mediated high stretch-induced modulation of cAMP and ATP synthesis inside ASMCs. Furthermore, such interactive miRNA-mRNA pairs may provide new insights for the discovery of effective biomarkers/therapeutic targets for the diagnosis and treatment of VILI and other MV-associated respiratory diseases.

Keywords: airway smooth muscle cells; high stretch; microRNA; purine metabolism; ventilator-induced lung injury.

Figure 1

Schematic diagram of the experimental protocol to identify high stretch-responsive events and the underlying regulatory miRNA–mRNA interactions in human airway smooth muscle cells (ASMCs). (A) The control and experimental ASMCs were cultured on an elastic membrane in either static (left) or high-stretch (right, 13% cyclic strain, 0.5 Hz) conditions, respectively. (B) Flow chart of high-stretch treatment, whole genome-wide RNA-Seq, bioinformatics analysis, and functional analysis to identify high stretch-induced enriched events and miRNA–mRNA interactions in cultured ASMCs.

Figure 2

Identification of target differentially expressed mRNAs (DE-mRNAs) in ASMCs cultured under static or high-stretch conditions. (A) Overlapped genes between targets of down-regulated DE-miRNAs and up-regulated DE-mRNAs (up panel) and overlapped genes between targets of up-regulated DE-miRNAs and down-regulated DE-mRNAs (down panel). (B) Volcano plot of 283 target DE-mRNAs. Black dots indicate the genes that were differently expressed in ASMCs cultured in high-stretch groups compared to static groups with p < 0.01 and |log₂FC| > 2. Red dots indicate the top 10 upregulated mRNAs and green dots indicate the top 10 down-regulated mRNAs.

Figure 3

Purine metabolism was enriched in the KEGG pathway and GO enrichment analysis of the 283 target DE-mRNAs. (A) Significant enrichment of the KEGG pathway of the 283 target DE-mRNAs. (B) Significant enrichment GO of the 283 target DE-mRNAs. Purple bars stand for the BP, and green bars stand for the MF. Box indicates that these events were related to purine metabolism. The circle indicates the number of enriched genes in each term.

Figure 4

Purine metabolism was enriched in the first module of the PPI network. (A) The PPI network construction and module analysis. The blue nodes represent the up-regulated target DE-mRNAs. The green nodes represent the down-regulated target DE-mRNAs. The size of the nodes indicates the degree of the target DE-mRNAs, and the lines represent the interaction between gene-encoded proteins. (B) Significant enrichment of the KEGG pathway of the 38 DE-mRNAs in the first module of the PPI network. Box indicates that these events were related to purine metabolism. (C) Significant enrichment GO of the 38 DE-mRNAs in the first module of the PPI network. Green bars stand for the BP, and red bars stand for the MF. Box indicates that these events were related to purine metabolism. The circle indicates the number of enriched genes in each term.

Figure 5

High stretch modulated purine metabolism. (A) The miRNA–mRNA interactions related to purine metabolism response to high stretch of ASMCs. The red and green colors represent up-regulation and down-regulation, respectively. The bar graph shows the relative cAMP (B) and ATP (C) levels inside ASMCs cultured in static or high-stretch conditions. Each experiment was repeated three times (n = 3). Data are presented as means ± S.E.M. * p < 0.05 and ** p < 0.01 compared to static groups.

Figure 6

miR-370-5p partially reversed high stretch-induced change in cAMP and ATP levels inside ASMCs cultured in static or high-stretch conditions. (A) The cellular activity of ASMCs treated with miR-370-5p mimics or inhibitor. (B) Relative expression levels of miR-370-5p in ASMCs treated with miR-370-5p mimics or inhibitor. (C,D) The mRNA expression of PDE4D, AK7 of ASMCs treated with miR-370-5p mimics or inhibitor, respectively. (E,F) The relative cAMP, ATP level inside ASMCs cultured with miR-370-5p mimics or inhibitor, respectively. (G,H) The relative cAMP, ATP level inside ASMCs cultured in static or high-stretch conditions with or without miR-370-5p mimics. Each experiment was repeated three times (n = 3). Data are presented as means ± S.E.M. Markers denote not significant (n.s) or significant difference from control conditions on NC mimics or static group (* p < 0.05), NC inhibitor (^# p < 0.05), high-stretch group with NC mimics (^$ p < 0.05), ** p < 0.01, ^## p < 0.01, ^$$ p < 0.01.