miR‑106b‑5p modulates acute pulmonary embolism via NOR1 in pulmonary artery smooth muscle cells

Abstract

Acute pulmonary embolism (APE) is a common cause of acute cardiovascular failure and has a high morbidity and mortality rate. Inhibiting the excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) is a potential treatment strategy following an APE. Various microRNAs (miRNAs/miRs) have been shown to regulate cell proliferation, apoptosis and other physiological processes. However, the specific mechanisms underlying the action of multiple miRNAs are still not understood in APE. In the present study, the role of miR‑106b‑5p on APE was demonstrated in platelet‑derived growth factor (PDGF)‑induced PASMCs in vitro and in an APE‑mouse model in vivo. The results showed that miR‑106b‑5p expression was downregulated in PDGF‑induced PASMCs and APE mice, and NOR1 levels were upregulated. Proliferating cell nuclear antigen (PCNA) expression levels in cells and proliferation of PASMCs proliferation and migration were reduced following treatment with miR‑106b‑5p agomiR, and increased following treatment with miR‑106b‑5p antagomiR. miR‑106b‑5p targeted the 3' untranslated region of NOR‑1 mRNA and reduced NOR1 expression. NOR1 overexpression reversed the effects of miR‑106‑5p on PDGF‑induced PASMCs. The functional roles of miR‑106b‑5p in PDGF‑induced PASMCs and an APE mouse‑model, and the underlying molecular mechanisms were evaluated. AgomiR‑106b‑5p improved APE‑induced mortality and pulmonary vascular proliferation in mice. These data suggest that miR‑106‑5p is a novel regulator of proliferation of PASMCs and of pulmonary vascular remodeling through PDGF‑induced PASMCs in an APE mouse model via targeting NOR1. These results expand the understanding of the pathogenesis underlying APE and highlight potential novel therapeutic targets.

Figure 1

miR-106b-5p and NOR-1-PCNA expression levels following PDGF treatment in PASMCs. (A) miR-106-5p levels, (B) NOR1 mRNA expression levels and (C) PCNA protein expression levels following treatment of PASMCs with 0, 10, 20 or 40 ng/ml PDGF for 24 h. n=4. ^*P<0.05 vs. Ctrl group. Ctrl, control; miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen.

Figure 2

AgomiR-106b-5p alleviates PDGF-induced proliferation and migration of PASMCs. PASMCs were treated with 20 ng/ml PDGF and subsequently treated with agomiR-106b-5p. (A) Cell viability was assessed using a Cell Counting Kit-8 assay. (B) Western blotting and densitometry analysis of PCNA expression. (C) Transwell invasion assays were performed to assess invasion of cells following the various treatments. Scale bars: 20 μm. ^*P<0.05 vs. Ctrl group. ^#P<0.05 vs. PDGF group. miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; NC, negative control; Ctrl, control.

Figure 3

AntagomiR-106b-5p increases proliferation and migration of PASMCs. (A) After treatment with PDGF or antagomiR-106b-5p, the viability of PASMCs was assessed using a Cell Counting Kit-8 assay. (B) PCNA protein expression following the various treatments. (C) Transwell invasion assays were performed to assess invasion following the various treatments. Scale bars: 20 μm. ^*P<0.05 vs. Ctrl group. ^#P<0.05 vs. PDGF group. miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; NC, negative control; Ctrl, control.

Figure 4

miR-106b-5p targeting of NOR-1 is confirmed using Ago2 immunoprecipitation and luciferase reporter assays. (A) Bioinformatics analysis using TargetScan, PicTar and miRanda predicted that NOR-1 was a potential target gene of miR-106b-5p. (B) PASMCs were transfected with 100 nM agomiR-106b-5p or agomiR-NC for 24 h, the cells were collected and immunoprecipitated using Ago2 or IgG and the fold-changes in NOR-1 transcript levels were calculated. ^*P<0.05 vs. agomiR-NC group. (C) After transfection with WT-NOR-1 or Mut-NOR-1 and treatment with agomiR-106b-5p or antagomiR-106b-5p in PASMCs, luciferase assays were performed to demonstrate miR-106b-5p directly targeted NOR-1. ^*P<0.05 vs. Ctrl group. (D) After treatment with agomiR-106b-5p, antagomiR-106b-5p or PDGF in PASMCs, the mRNA expression levels of NOR-1 were measured. ^*P<0.05 vs. Ctrl group. ^#P<0.05 vs. PDGF group. miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell; NC, negative control; WT, wild-type; Mut, mutated; Ctrl, control.

Figure 5

NOR-1 activation improves the inhibitory action of miR-106b-5p on the proliferation and migration of PASMCs. After overexpression of NOR-1 in PASMCs, cells were treated with agomiR-106b-5p and/or PDGF. (A) Cell Counting Kit-8 assays were used to assess cell viability. ^*P<0.05 vs. Ctrl group. ^#P<0.05 vs. PDGF group. ^&P<0.05 vs. NOR1 group. ^$P<0.05 vs. NOR1 + agomiR-106b-5p group. (B) Transwell invasion assays were used to assess invasion of cells. Scale bars: 20 μm. ^*P<0.05 vs. agomiR-106b-5p group. (C) NOR-1 and PCNA protein expression levels were measured using western blotting. ^*P<0.05 vs. agomiR-106b-5p group. miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell.

Figure 6

AgomiR-106b-5p alleviates APE-induced lung injury through inhibition of NOR-1 in a mouse model of APE. After treatment with agomiR-106b-5p in the APE mouse model, (A) survival was monitored for 15 days (n=10). (B) Pulmonary vascular proliferation was assessed by measuring the mRNA expression levels of NOR-1 and PCNA protein expression levels in the pulmonary artery and (C) immunohistochemistry analysis of NOR-1 was performed in the pulmonary vasculature after 7 days, n=6. Scale bars: 20 μm. n=10. ^*P<0.05 vs. Ctrl; ^#P<0.05 vs. APE group. Ctrl, control; miR, microRNA; PDGF, platelet-derived growth factor; PASMC, pulmonary artery smooth muscle cell; APE, acute pulmonary embolism.