Deregulated microRNA and mRNA expression profiles in the peripheral blood of patients with Marfan syndrome

Abstract

Background: MicroRNAs (miRNAs) are small RNAs regulating gene expression post-transcriptionally. While acquired changes of miRNA and mRNA profiles in cancer have been extensively studied, little is known about expression changes of circulating miRNAs and messenger RNAs (mRNA) in monogenic constitutional anomalies affecting several organ systems, like Marfan syndrome (MFS). We performed integrated miRNA and mRNA expression profiling in blood samples of Marfan patients in order to investigate deregulated miRNA and mRNA networks in these patients which could serve as potential diagnostic and prognostic tools for MFS therapy.

Methods: MiRNA and mRNA expression profiles were determined in blood samples from MFS patients (n = 7) and from healthy volunteer controls (n = 7) by microarray analysis. Enrichment analyses of altered mRNA expression were identified using bioinformatic tools.

Results: A total of 28 miRNAs and 32 mRNAs were found to be significantly altered in MFS patients compared to controls (> 2.0-fold change, adjusted P < 0.05). The expression of 11 miRNA and 6 mRNA candidates was validated by RT-qPCR in an independent cohort of 26 MFS patients and 26 matched HV controls. Significant inverse correlations were evident between 8 miRNAs and 5 mRNAs involved in vascular pathology, inflammation and telomerase regulation. Significant positive correlations were present for 7 miRNAs with age, for 2 miRNAs with the MFS aortic root status (Z-score) and for 7 miRNAs with left ventricular end-diastolic diameter in MFS patients. In addition, miR-331-3p was significantly up-regulated in MFS patients without mitral valve prolapse (MVP) as compared with patients with MVP.

Conclusions: Our data show deregulated gene and miRNA expression profiles in the peripheral blood of MFS patients, demonstrating several candidates for prognostic biomarkers for cardiovascular manifestations in MFS as well as targets for novel therapeutic approaches. A deregulation of miRNA expression seems to play an important role in MFS, highlighting the plethora of effects on post-transcriptional regulation of miRNAs and mRNAs initiated by constitutional mutations in single genes. Trial registration Nr: EA2/131/10 . Registered 28 December, 2010.

Keywords: Fibrillin; Integration analysis; Marfan syndrome; MicroRNA; mRNA.

Fig. 1

Unsupervised hierarchical clustering (Euclidian distance, complete linkage) of the 14 samples based on expression of the 63 with significant highest variance out of the 1205 miRNAs. The heatmap shows miRNAs with high expression in red, miRNAs with low expression in green. The red lines indicate three main clusters of samples

Fig. 2

Unsupervised hierarchical clustering (Euclidian distance, complete linkage) of the 14 samples based on expression of the 65 transcripts with the highest expression variances out of the 50,599 biological features. The heatmap shows transcripts with high expression in red, transcript with low expression in green. The red lines indicate three main clusters of samples

Fig. 3

Validation of differentially expressed miRNAs in the blood of MFS patients (n = 26) compared to HV controls (n = 26) as determined by RT-qPCR (P < 0.05). Mean ΔCt MFS and HV controls (Lower ΔCt, higher expression level). RNAU6B as an endogenous control for normalization, Unpaired-two-tailed t tests and ± standard deviation (STDV) were used to evaluate differences in expression. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 4

Validation of differentially expressed mRNAs in the blood of MFS patients (n = 26) compared to HV controls (n = 26) as determined by RT-qPCR (P < 0.05). Mean ΔCt MFS and HV controls (Lower ΔCt, higher expression level). β-Actin as an endogenous housekeeping gene for normalization, Unpaired-two-tailed t tests and ± standard deviation (STDV) were used to evaluate differences in expression. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001