MiRNAs in Systemic Sclerosis Patients with Pulmonary Arterial Hypertension: Markers and Effectors

Abstract

Background: Pulmonary arterial hypertension (PAH) is a major cause of death in systemic sclerosis (SSc). Early detection may improve patient outcomes.

Methods: We searched for circulating miRNAs that would constitute biomarkers in SSc patients with PAH (SSc-PAH). We compared miRNA levels and laboratory parameters while evaluating miRNA levels in white blood cells (WBCs) and myofibroblasts.

Results: Our study found: 1) miR-26 and miR-let-7d levels were significantly lower in SSc-PAH (n = 12) versus SSc without PAH (SSc-noPAH) patients (n = 25); 2) a positive correlation between miR-26 and miR-let-7d and complement-C3; 3) GO-annotations of genes that are miR-26/miR-let-7d targets and that are expressed in myofibroblast cells, suggesting that these miRNAs regulate the TGF-β-pathway; 4) reduced levels of both miRNAs accompanied fibroblast differentiation to myofibroblasts, while macitentan (endothelin receptor-antagonist) increased the levels. WBCs of SSc-noPAH and SSc-PAH patients contained equal amounts of miR-26/miR-let-7d. During the study, an echocardiograph that predicted PAH development, showed increased pulmonary artery pressure in three SSc-noPAH patients. At study initiation, those patients and an additional SSc-noPAH patient, who eventually developed PAH, had miR-let-7d/miR-26 levels similar to those of SSc-PAH patients. This implies that reduced miR-let-7d/miR-26 levels might be an early indication of PAH.

Conclusions: miR-26 and miR-let-7d may be serological markers for SSc-PAH. The results of our study suggest their involvement in myofibroblast differentiation and complement pathway activation, both of which are active in PAH development.

Keywords: biomarkers; complement; macitentan; miRNA; myofibroblasts; pulmonary arterial hypertension (PAH); systemic sclerosis.

Figure 1

Median miRNA levels in the plasma of SSc patients with and without PAH. RNA was isolated from plasma of 25 SSc-noPAH and 12 SSc-PAH patients. The levels of seven miRNAs were evaluated by qRT-PCR. miR-16 served for normalization. The boxplot presents five values (minimum: (Q1–1.5*IQR); first quartile (Q1/25th percentile); median; third quartile (Q3/75th percentile); and maximum (Q3 + 1.5*IQR)) of the miRNAs. miR: miRNA; PAH: Pulmonary arterial hypertension; SSc-noPAH: Patients with SSc without PAH; SSc-PAH: Patients with SSc and PAH.

Figure 2

Mean miRNA levels in the SSc-noPAH group. miRNA levels in the plasma of 25 SSc-noPAH patients were evaluated using qRT-PCR. miR-16 served for normalization. Patients underwent echocardiography before blood sampling and at the end of the study. miRNA values of the patients with stable echo results during this time are marked in full black dots (n = 21). The miRNA levels of patients whose echo levels increased during this period are marked by empty circles (n = 4). All y-axis values are presented relative to miR-16. The red line indicates the mean value of each group.

Figure 3

miR-26 and miR-let-7d levels in WBCs of SSc-noPAH and SSc-PAH patients. WBCs were isolated from 7 SSc-PAH and 8 SSc-noPAH patients. RNA was isolated from the cells and the levels of miR-26 and miR-let-7d were evaluated with qRT-PCR. miR-16 served as the normalizing miRNA. The graph represents miRNA expression levels relative to miR-16. Horizontal bars represent the mean values of the miRNAs.

Figure 4

TGF-β induced myofibroblast differentiation is accompanied by reduced miR-26 and miR-let-7d levels. Dermal fibroblasts were isolated from healthy patients undergoing plastic surgery. The cells were exposed to TGF-β (10 ng/mL) for 48 h or 72 h. Then the cells were either harvested and counted (72 h) or harvested and their proteins and RNA isolated (48 h). The levels of the myofibroblast markers αSMA (A) and collagen I (B) were assessed by western blot, and the levels of miR-21, miR-26 and miR-let-7d were assessed with qRT-PCR (D). (C) is a representative blot of αSMA and collagen in cells that were treated or not treated with TGF-β (n = 4). Columns represent mean and whiskers represent standard errors. *—Statistically significant results, p ≤ 0.05.