Circulating plasma microRNAs in systemic sclerosis-associated pulmonary arterial hypertension

Abstract

Objectives: SSc-associated pulmonary arterial hypertension (SSc-APAH) is a late but devastating complication of SSc. Early identification of SSc-APAH may improve survival. We examined the role of circulating miRNAs in SSc-APAH.

Methods: Using quantitative RT-PCR the abundance of mature miRNAs in plasma was determined in 85 female patients with ACA-positive lcSSc. Twenty-two of the patients had SSc-APAH. Sixty-three SSc controls without PAH were matched for disease duration. Forty-six selected miRNA plasma levels were correlated with clinical data. Longitudinal samples were analysed from 14 SSc-APAH and 27 SSc patients.

Results: The disease duration was 12 years for the SSc-APAH patients and 12.7 years for the SSc controls. Plasma expression levels of 11 miRNAs were lower in patients with SSc-APAH. Four miRNAs displayed higher plasma levels in SSc-APAH patients compared with SSc controls. There was significant difference between groups for miR-20a-5p and miR-203a-3p when correcting for multiple comparisons (P = 0.002 for both). Receiver operating characteristics curve showed AUC = 0.69-0.83 for miR-21-5p and miR-20a-5p or their combination. miR-20a-5p and miR-203a-3p correlated inversely with NT-pro-Brain Natriuretic Protein levels (r = -0.42 and -0.47). Mixed effect model analysis could not identify any miRNAs as predictor of PAH development. However, miR-20a-5p plasma levels were lower in the longitudinal samples of SSc-APAH patients than in the SSc controls.

Conclusions: Our study links expression levels of the circulating plasma miRNAs, especially miR-20a-5p and miR-203a-3p, to the occurrence of SSc-APAH in female patients with ACA-positive lcSSc.

Keywords: NT-pro-Brain Natriuretic Protein; microRNA; pulmonary arterial hypertension; systemic sclerosis.

Fig. 1

miRNA expressions in the plasma of SSc patients with APAH or without Displayed are −ΔCq values of miRNAs in plasma of patients with SSc or with SSc-APAH. Shown are the raw data (triangles), median (line) and interquartile range (box). SSc-APAH: SSc-associated pulmonary arterial hypertension; Cq: quantitation cycle.

Fig. 2

AUC analysis of plasma miRNA levels to predict pulmonary arterial hypertension in SSc patients AUC values are depicted for miR-20a-5p (blue), miR-21-5p (red), combination of the miR-20a-5p and miR-21-5p (black). AUC: area under the curve.

Fig. 3

Correlations between plasma miR-20a-5p and miR-203a-3p expressions and serum NT-pro-BNP Displayed are correlation between –ΔCq values of miRNA miR-20a-5p (A) and miR-203a-3p (B) in plasma and serum NT-pro-BNP levels (log10 transformed). SSc patients with APAH (n = 19) are depicted as red triangles and SSc patients without APAH (n = 41) are shown as blue circles. NT-pro-BNP: NT-pro-Brain Natriuretic Protein; Cq: quantitation cycle; APAH: associated pulmonary arterial hypertension.

Fig. 4

Plasma expression levels of miR-20a-5p over time in SSc patients with APAH or SSc controls Samples from patients that develop SSc-APAH later are depicted as red triangles (n = 14, SSc-APAH). Samples from patients that did not develop SSc-APAH are shown as blue circles (n = 27, SSc). Two to three samples/patient were analysed. (A) Student’s t-test analysis of the bulk of all longitudinal samples for the two groups are shown. (B) The longitudinal distribution of the samples is shown in relation of the time before the last sample (of the cross-sectional analysis) was taken. Expression levels of miR-20a-5p were analysed by linear generalized estimating equations taking in to account the repeated samples of the individuals. SSc-APAH: SSc-associated pulmonary arterial hypertension; Cq: quantitation cycle.