miR-30d Attenuates Pulmonary Arterial Hypertension via Targeting MTDH and PDE5A and Modulates the Beneficial Effect of Sildenafil

Abstract

Pulmonary arterial hypertension (PAH) is associated with aberrant pulmonary vascular smooth muscle cell (PASMC) function and vascular remodeling. MiR-30d plays an important role in the pathogenesis of several cardiovascular disorders. However, the function of miR-30d in PAH progression remained unknown. Our study shows that circulating miR-30d level is significantly reduced in the plasma from PAH patients. In miR-30d transgenic (TG) rats, overexpressing miR-30d attenuates monocrotaline (MCT)-induced pulmonary hypertension (PH) and pulmonary vascular remodeling. Increasing miR-30d also inhibits platelet-derived growth factor-bb (PDGF-bb)-induced proliferation and migration of human PASMC. Metadherin (MTDH) and phosphodiesterase 5A (PDE5A) are identified as direct target genes of miR-30d. Meanwhile, nuclear respiratory factor 1 (NRF1) acts as a positive upstream regulator of miR-30d. Using miR-30d knockout (KO) rats treated with sildenafil, a PDE5A inhibitor that is used in clinical PAH therapies, it is further found that suppressing miR-30d partially attenuates the beneficial effect of sildenafil against MCT-induced PH and vascular remodeling. The present study shows a protective effect of miR-30d against PAH and pulmonary vascular remodeling through targeting MTDH and PDE5A and reveals that miR-30d modulates the beneficial effect of sildenafil in treating PAH. MiR-30d should be a prospective target to treat PAH and pulmonary vascular remodeling.

Keywords: MTDH; PDE5A; miR‐30d; pulmonary arterial hypertension; pulmonary arterial smooth muscle cell; sildenafil.

Figure 1

miR‐30d overexpression attenuates pulmonary hypertension and vascular remodeling. (A and B) Relative miR‐30d expression level in plasma samples (A) and in plasma‐derived extracellular vesicles (EVs) (B) from patients with idiopathic pulmonary arterial hypertension (PAH) versus healthy controls (n = 10 vs 8). C) Relative miR‐30d expression level in lung tissues of rats with monocrotaline (MCT)‐induced pulmonary hypertension (PH) (n = 10‐13). (D and E) Right ventricular systolic pressure (RVSP) (D) and Fulton index (RV/(LV+S) ratio) (E) were assessed (n = 6‐7). F) Immunohistochemical staining with α‐SMA antibody for analysis of the number of muscularized distal pulmonary arteries (10–50 µm in diameter, scale bar = 20 µm) and the medial wall thickness of pulmonary arteries (50–150 µm in diameter, scale bar = 100 µm) (n = 5‐6). G) qRT‐PCR for CDK1, CDK6, and CCNA2 mRNA levels in lung tissues (n = 6). H) Western blot for CDK6 in lung tissues (n = 6). Data are shown as means ± SD. Data between 2 groups were compared by Mann‐Whitney U test for (A) and by independent‐sample two‐tailed Student's t‐test for (B) and (C). Data among 4 groups were compared by robust two‐way ANOVA test followed by post‐hoc pairwiseMedianTest using the rcompanion package for CCNA2 in (G), and by two‐way ANOVA test followed by Tukey post hoc test for data in other figures. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Overexpressing miR‐30d inhibits hPASMC proliferation and migration. A) Relative miR‐30d expression level in human pulmonary arterial smooth muscle cells (hPASMC) stressed with platelet‐derived growth factor‐bb (PDGF‐bb) for 48h (n = 6). B) Relative miR‐30d expression level in extracellular vesicles (EVs) isolated from the culture medium of rat PASMC after treatment with PDGF‐bb or control vehicles for 48h (n = 6). C) The overexpression efficiency of miR‐30d mimic in hPASMC (n = 3). D) Representative images and quantification of EdU/Hoechst staining of miR‐30d mimic or negative control (NC) transfected hPASMC under PDGF‐bb stress (n = 5). Scale bar = 200 µm. E) Scratch wound healing assay was performed to investigate the effect of miR‐30d mimic on hPASMC migration. Representative images and relative wound closure of miR‐30d mimic or NC transfected hPASMC under PDGF‐bb stress (n = 3). Scale bar = 200 µm. F) qRT‐PCR for CDK1, CDK6, and CCNA2 mRNA levels in hPASMC (n = 6). G) Western blot for PCNA and NFATC4 in hPASMC (n = 3). Data are shown as means ± SD. Data between 2 groups were compared by independent‐sample two‐tailed Student's t‐test for (A) to (C). Data among 4 groups were compared by two‐way ANOVA test followed by Tukey post hoc test for (D) to (G). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

MTDH and PDE5A are downstream targets of miR‐30d. A) The intersection of TargetScan, MicroT‐CDS, and miRDB for predicting target genes of miR‐30d. B) KEGG pathway of the enriched potential target genes of miR‐30d. (C and D) Western blot for MTDH (C) and PDE5A (D) in human pulmonary arterial smooth muscle cells (hPASMC) transfected with miR‐30d mimic or inhibitor and negative control (NC) (n = 3). E) Luciferase reporter assay was performed in hPASMC treated with platelet‐derived growth factor‐bb (PDGF‐bb), simultaneously transfected with the recombinant plasmids containing wild type 3’‐UTR region of MTDH or PDE5A or relevant mutant sequence along with miR‐30d mimic or NC mimic (n = 6). F) Western blot for MTDH and PDE5A in lung tissues from wild type (WT) or miR‐30d transgenic (TG) rats injected with monocrotaline (MCT) or control (n = 6). Data are shown as means ± SD. Data between 2 groups were compared by independent‐sample two‐tailed Student's t‐test for (C) and (D). Data among 4 groups were compared by two‐way ANOVA test followed by Tukey post hoc test for (E) and (F). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

miR‐30d inhibits hPASMC proliferation and migration through targeting MTDH and PDE5A. A and C) EdU‐positive cells in MTDH (A) or PDE5A (C) overexpressing and/or miR‐30d overexpressing human pulmonary arterial smooth muscle cells (hPASMC) under PDGF‐bb stress (n = 5‐6). Scale bar = 200 µm. B and D) Wound closure of scratch experiment in MTDH (B) or PDE5A (D) overexpressing and/or miR‐30d overexpressing hPASMC under PDGF‐bb stress (n = 3). Scale bar = 200 µm. Data are shown as means ± SD. Data among 4 groups were compared by two‐way ANOVA test followed by Tukey post hoc test for (A) to (D). **P < 0.01; ***P < 0.001.

Figure 5

NRF‐1 is upstream regulator of miR‐30d. A) Predicting NRF1 as potential upstream regulators of miR‐30d through DIANA TOOLS (miRGen v.3). B) According to the online single‐cell RNA sequencing data (GEO: GSE210248) of the isolated pulmonary arteries from idiopathic pulmonary arterial hypertension (PAH) patient lungs versus donor lungs, comparison of NRF1 expressions in the cluster of pulmonary arterial smooth cells (PASMC) was demonstrated. C) qRT‐PCR analysis for NRF1 in lung tissues from monocrotaline (MCT)‐induced pulmonary hypertension (PH) model and in PDGF‐treated human PASMC (n = 6). D and E) Western blot for NRF1 in lung tissues from MCT‐induced PH model (D) and in PDGF‐treated human PASMC (E) (n = 6). F) qRT‐PCR for NRF1 and miR‐30d in human PASMC transfected with NRF1 siRNA (si‐NRF1) or negative control (NC‐siRNA) (n = 6). G) ChIP assay to determine the enrichment of NRF1 in the promoter region of miR‐30d using rat PASMC (n = 3). H) EdU‐positive cells in human PASMC co‐transfected with miR‐30d mimic and/or si‐NRF1 under PDGF‐bb stress (n = 5). Scale bar = 200 µm. I) Wound closure of scratch experiment in human PASMC co‐transfected with miR‐30d mimic and/or si‐NRF1 under PDGF‐bb stress (n = 3). Scale bar = 200 µm. Data are shown as means ± SD. Data between 2 groups were compared by independent‐sample two‐tailed Student's t‐test for (C) to (E) and (G), and for miR‐30d in (F), and by Mann‐Whitney U test for NRF‐1 in (F). Data among 4 groups were compared by two‐way ANOVA test followed by Tukey post hoc test for (H) and (I). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

MiR‐30d modulates the beneficial effect of sildenafil against pulmonary hypertension. We utilized miR‐30d knockout (KO) rats to receive sildenafil treatment in a monocrotaline (MCT)‐induced pulmonary hypertension (PH) model. A and B) Right ventricular systolic pressure (RVSP) (A) and Fulton index (RV/(LV+S) ratio) (B) were assessed (n = 5‐7 for control‐treated MCT rats, n = 8 for sildenafil‐treated MCT rats). C) Immunohistochemical staining with α‐SMA antibody for analysis of the number of muscularized distal pulmonary arteries (10–50 µm in diameter, scale bar = 20 µm) and the medial wall thickness of pulmonary arteries (50–150 µm in diameter, scale bar = 100 µm) (n = 4‐5). D) qRT‐PCR for CDK1, CDK6, and CCNA2 mRNA levels in lung tissues (n = 5‐7 for control‐treated MCT rats, n = 8 for sildenafil‐treated MCT rats). E) Western blot for MTDH and PDE5A protein levels in lung tissues of WT or miR‐30d KO rats in PH model treated with sildenafil or not (n = 6). Data are shown as means ± SD. Data among 4 groups were compared by robust two‐way ANOVA test followed by post‐hoc pairwiseMedianTest using the rcompanion package for medial wall thickness in (C) and for CDK6 in (D), and by two‐way ANOVA test followed by Tukey post hoc test for data in other figures. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Schematic diagram illustrating the role of miR‐30d in pulmonary arterial hypertension. MiR‐30d is downregulated in pulmonary arterial hypertension (PAH) which is mainly induced by a reduced expression of NRF1. Overexpressing miR‐30d prevents PAH and pulmonary vascular remodeling and inhibits pulmonary arterial smooth muscle cell (PASMC) proliferation and migration through directly targeting metadherin (MTDH) and phosphodiesterase 5A (PDE5A). MiR‐30d, at least in part, contributes to the effect of sildenafil in treating PAH.