Phosphatidylinositol 3-Kinase-DNA Methyltransferase 1-miR-1281-Histone Deacetylase 4 Regulatory Axis Mediates Platelet-Derived Growth Factor-Induced Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells

Abstract

Background: Platelet-derived growth factor BB, a potent mitogen of pulmonary artery smooth muscle cells (PASMCs), has been implicated in pulmonary arterial remodeling, which is a key pathogenic feature of pulmonary arterial hypertension. Previous microRNA profiling in platelet-derived growth factor BB-treated PASMCs found a significantly downregulated microRNA, miR-1281, but it has not been associated with any cellular function, and we investigated the possibility.

Methods and results: Real-time quantitative reverse transcription-polymerase chain reaction assay proved that downregulation of miR-1281 was a conserved phenomenon in human and rat PASMCs. Overexpression and inhibition of miR-1281 in PASMCs promoted and suppressed, respectively, the cell proliferation and migration. Bioinformatic prediction and 3'-untranslated region reporter assay identified histone deacetylase 4 to be a direct target of miR-1281. Supporting this, proliferation and migration assay demonstrated the cellular function of histone deacetylase 4 is inversely correlated with that of miR-1281. Mechanistically, it is found that platelet-derived growth factor BB activates the phosphatidylinositol 3-kinase pathway, which then induces the expression of DNA methyltransferase 1, leading to enhanced methylation of a flanking CpG island and repressed miR-1281 expression. Finally, a reduced miR-1281 level was consistently identified in hypoxic PASMCs in vitro, in pulmonary arteries of rats with monocrotaline-induced pulmonary arterial hypertension, and in serum of patients with coronary heart disease-pulmonary arterial hypertension. These data suggest that there may be a diagnostic and therapeutic use for miR-1281.

Conclusions: Herein, we report a novel regulatory axis, phosphatidylinositol 3-kinase-DNA methyltransferase 1-miR-1281-histone deacetylase 4, integrating multiple epigenetic regulators that participate in platelet-derived growth factor BB-stimulated PASMC proliferation and migration and pulmonary vascular remodeling.

Keywords: DNA methyltransferase 1; epigenetics; histone deacetylase 4; hypertension; miRNA; platelet‐derived growth factor; pulmonary; pulmonary arterial smooth muscle cells; vascular remodeling; vascular smooth muscle.

Figure 1

miR‐1281 is downregulated in platelet‐derived growth factor (PDGF) BB–stimulated pulmonary artery smooth muscle cells (PASMCs). A, Sequence of human miR‐1281 precursor was aligned with corresponding sequences 5′ upstream of gene EP300 in rat and mouse genomes. Red box indicates the region generating mature miR‐1281 (underlined). B, Real‐time quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) measurements of miR‐1281 level in human and rat PASMCs (HPASMCs and RPASMCs, respectively) treated with dimethyl sulfoxide (DMSO; ‐PDGFBB) or PDGFBB. C, Time‐course assay of PDGFBB effect on miR‐1281 level in rat PASMCs. Significant difference was analyzed for each of 2 neighboring time points and marked by asterisks and broken lines. D, Dosage‐course assay of PDGFBB effect on miR‐1281 level in rat PASMCs. Significant differences were analyzed for each of 2 neighboring dosage points and as broken line indicated. E, Effects of different growth factors on PASMC miR‐1281 expression were evaluated by qRT‐PCR. DMSO treatment was used as negative control. Angiotensin 2 (Ang2), transforming growth factor‐β (TGF‐β), insulin‐like growth factor (IGF), vascular endothelial growth factor (VEGF), endothelin 1 (ET‐1), PDGFAA, and fibroblast growth factor (FGF) were applied at concentrations as indicated. For this and all subsequent qRT‐PCR data: miRNA levels are normalized with small nucleolar 202 or small nucleolar 44 for rat or human samples, respectively, and mRNA levels are normalized with β‐actin. Fold changes are calculated by comparing each normalized measurement with normalized corresponding control. Data are expressed as means±SD, verified by at least 3 biological replicates. *P<0.05, **P<0.01, ***P<0.001 compared with control or as specified by broken lines in respective figures. Exact P values in consecutive order are: 0.000599, 0.000926, 0.016403, 0.044714, 0.030503,0.040582, 0.047031, 0.001946, 0.009166, 0.033907, 0.002544, 0.000013.

Figure 2

miR‐1281 suppresses pulmonary artery smooth muscle cell (PASMC) proliferation and migration. A, Real‐time quantitative reverse transcription–polymerase chain reaction (qRT‐PCR) analysis of overexpression level of miR‐1281 in PASMCs achieved by transfection of miR‐1281 mimic. B, Representative figures of EdU assay and statistical analysis showing that miR‐1281 mimic inhibited PASMC proliferation under culturing conditions without and with platelet‐derived growth factor (PDGF) BB treatments. C, qRT‐PCR analysis of inhibitory efficiency of miR‐1281 inhibitor in PASMCs. D, Representative figures of EdU assay and statistical analysis showing that miR‐1281 inhibitor promoted PASMC proliferation. E, Representative figures of wound‐healing assay and statistical analysis showing that miR‐1281 mimic inhibited PASMC migration under culturing conditions without and with PDGFBB treatments. F, Representative figures of wound‐healing assay and statistical analysis showing that miR‐1281 inhibitor promoted PASMC migration. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; and NC, negative control. *P<0.05, **P<0.01, ***P<0.001 compared with control or as specified by broken lines in respective figures. Exact P values in consecutive order are: 0.00045, 0.012642, 0.001155, 0.001081, 0.009504,0.03544, 0.003835, 0.002613.

Figure 3

Histone deacetylase 4 (HDAC4) is a direct target gene of miR‐1281 in pulmonary artery smooth muscle cells (PASMCs). A, Alignment of miR‐1281 with its putative binding sites in 3′‐untranslated regions (UTRs) of protein tyrosine phosphate, recepter type, D (PTPRD), HDAC4, and wingless‐type MMTV integration site family member 3A (WNT3A) (3′‐UTR–wild type [WT]), and with corresponding mutated 3′‐UTR binding sites (3′‐UTR Mut). B, Respective 3′‐UTR–WT construct was cotransfected with either miR‐1281 mimic or control mimic into human embryonic kidney 293A cells, and luciferase reporter assay was performed to assess interactions between miR‐1281 and 3′‐UTRs of PTPRD, HDAC4, and WNT3A. C, Abolishing the interaction by cotransfecting miR‐1281 mimic with HDAC4 3′‐UTR Mut recovered miR‐1281 inhibition of luciferase expression. D, mRNA level of HDAC4 stayed unchanged in platelet‐derived growth factor (PDGF) BB–treated PASMCs. E, Protein level of HDAC4 increased in PDGFBB‐treated PASMCs. (F) Protein level of HDAC4 was downregulated by miR‐1281 mimic in PASMCs in the absence or presence of PDGFBB. β‐Actin was used as loading control. N.S. indicates not significant. *P<0.05 compared with control mimic. Exact P values in consecutive order are: 0.011313, 0.010948, 0.011313.

Figure 4

miR‐1281/histone deacetylase 4 (HDAC4) pathway regulates the transcription of multiple related transcription factors. A through C, A set of real‐time quantitative reverse transcription–polymerase chain reaction analyses exploring the possible regulatory network downstream of HDAC4 and miR‐1281. RNA samples were obtained from pulmonary artery smooth muscle cells transfected with small interfering (si)‐HDAC4, miR‐1281 mimic, miR‐1281 inhibitor, and their corresponding negative controls (NCs). CHOP indicates CCAAT/enhancer‐binding protein‐homologous protein; GLUT‐1, glucose transporter‐1; KLF, kruppel‐like factor; SMAD3, drosophila mothers against decapentaplegic protein 3; TRB3, tribbles homolog 3; and VEGF, vascular endothelial growth factor. *P<0.05, **P<0.01, ***P<0.001 compared with si‐NC or mimic or inhibitor control as broken lines indicated in each figure. Exact P values in consecutive order are: 0.045223, 0.021835, 0.009286, 0.042967, 0.020433, 0.019755, 0.000044, 0.037415, 0.034118, 0.029028, 0.024684, 0.001313, 0.000403, 0.045261.

Figure 5

Platelet‐derived growth factor (PDGF) BB–phosphatidylinositol 3‐kinase–DNA methyltransferase 1 (DNMT1) activation cassette mediates PDGFBB repression of miR‐1281. A, Expression of DNMT1 protein increased along with the increase of PDGFBB use. B, Real‐time quantitative reverse transcription–polymerase chain reaction analysis of small interfering (si)‐DNMT1 silencing efficiency in pulmonary artery smooth muscle cells (PASMCs) without and with PDGFBB treatments. C, Transfection of si‐DNMT1 recovered PDGFBB repression of miR‐1281 in PASMCs. D, Effects of different pathway inhibitors on DNMT1 expression were analyzed, and pictilisib was identified to inhibit PDGFBB upregulated mRNA level of DNMT1. E, Pictilisib inhibited PDGFBB upregulation of DNMT1 in a dose‐dependent manner. F, Pictilisib also inhibited PDGFBB‐repressed expression of miR‐1281. DMSO indicates dimethyl sulfoxide; NC, negative control; and N.S., not significant. *P<0.05, **P<0.01, ***P<0.001 compared with control or as specified by broken lines in respective figures. Exact P values in consecutive order are: 0.034235, 0.009507, 0.043504, 0.003449, 0.010124, 0.049599, 0.000249, 0.010004, 0.036281.

Figure 6

Reduced miR‐1281 level is identified in hypoxic pulmonary artery smooth muscle cells (PASMCs), rats with pulmonary arterial hypertension (PAH), and patients with PAH. The relative miR‐1281 levels were estimated by real‐time quantitative reverse transcription–polymerase chain reaction in PASMCs exposed to normoxia (21% O₂) or hypoxia (3% O₂) for 12 and 24 hours (A), pulmonary arteries individually collected from 4 rats with monocrotaline (MCT)–induced PAH and 4 controls (rats received intraperitoneal injections of normal saline; B), and serum individually collected from 13 healthy participants and 29 patients with coronary heart disease (CHD)–PAH (C). D, Proposed regulatory model of phosphatidylinositol 3‐kinase (PI3K)–DNA methyltransferase 1 (DNMT1)–miR‐1281–histone deacetylase 4 (HDAC4) axis. Upward arrow indicates upregulation or activation. Downward arrow indicates downregulation. N.S. indicates not significant; PDGF, platelet‐derived growth factor; and TF, transcription factor. *P<0.05, **P<0.01 compared with normoxia, saline or normal control. Exact P values in consecutive order are: 0.00332, 0.001079, 0.024024.