Microrna-486-5P Regulates Human Pulmonary Artery Smooth Muscle Cell Migration via Endothelin-1

Abstract

Pulmonary arterial hypertension (PAH) is a fatal or life-threatening disorder characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance. Abnormal vascular remodeling, including the proliferation and phenotypic modulation of pulmonary artery smooth muscle cells (PASMCs), represents the most critical pathological change during PAH development. Previous studies showed that miR-486 could reduce apoptosis in different cells; however, the role of miR-486 in PAH development or HPASMC proliferation and migration remains unclear. After 6 h of hypoxia treatment, miR-486-5p was significantly upregulated in HPASMCs. We found that miR-486-5p could upregulate the expression and secretion of ET-1. Furthermore, transfection with a miR-486-5p mimic could induce HPASMC proliferation and migration. We also found that miRNA-486-5p could downregulate the expression of SMAD2 and the phosphorylation of SMAD3. According to previous studies, the loss of SMAD3 may play an important role in miRNA-486-5p-induced HPASMC proliferation. Although the role of miRNA-486-5p in PAH in in vivo models still requires further investigation and confirmation, our findings show the potential roles and effects of miR-486-5p during PAH development.

Keywords: miR-486-5p; pulmonary arterial hypertension; pulmonary artery smooth muscle cells.

Figure 1

miR-486-5p increased the expression and secretion of ET-1 in HPASMCs. (A) After 6, 16, and 24 h of hypoxia treatment, the level of miR-486-5p was evaluated by qRT-PCR. HPASMCs were transfected with a miR-486-5p mimic or NC mimic, and the total RNA, cell lysates, and medium samples were collected after 48 h. (B) mRNA expression of ET-1 was determined by qRT-PCR. (C) ET-1 protein expression and (D) ET-1 secretion were determined by ELISA. Results are expressed as the means ± SEMs (n ≥3). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the T0 or NC mimic group.

Figure 2

miR-486-5p induced HPAMC proliferation and migration. (A) HPASMCs were transfected with the miR-486-5p mimic or NC mimic, and cell viability at 24, 48, and 72 h was determined with an MTT assay (B,C). The migration ability of HPASMCs was determined with a wound-healing assay. After creating wound gaps, cells were incubated for 6 h, and the cell migration rate was determined by calculating the distance migrated by the cells into the wound area. Scale bar: 200 μm. Results are expressed as the means ± SEMs (n ≥3). * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the NC mimic group.

Figure 3

miR-486-5p downregulated Smad2 and Smad3 in HPASMCs. (A) HPASMCs were transfected with the miR-486-5p mimic or NC mimic, and the total mRNA and cell lysate were collected after 48 h. The protein expression and phosphorylation of Smad2 and Smad3 were determined with Western blots. (B) The intensities of p-Smad2, p-Smad3, Smad2, Smad3, and β-actin were quantified using the ImageJ software. (C) The mRNA level of Smad2 was determined by qRT-PCR. Results are expressed as the means ± SEMs (n = 4). * p < 0.05 compared with the NC mimic group.