Differentially Expressed Plasma MicroRNAs and the Potential Regulatory Function of Let-7b in Chronic Thromboembolic Pulmonary Hypertension

Abstract

Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive disease characterized by misguided thrombolysis and remodeling of pulmonary arteries. MicroRNAs are small non-coding RNAs involved in multiple cell processes and functions. During CTEPH, circulating microRNA profile endued with characteristics of diseased cells could be identified as a biomarker, and might help in recognition of pathogenesis. Thus, in this study, we compared the differentially expressed microRNAs in plasma of CTEPH patients and healthy controls and investigated their potential functions. Microarray was used to identify microRNA expression profile and qRT-PCR for validation. The targets of differentially expressed microRNAs were identified in silico, and the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway database were used for functional investigation of target gene profile. Targets of let-7b were validated by fluorescence reporter assay. Protein expression of target genes was determined by ELISA or western blotting. Cell migration was evaluated by wound healing assay. The results showed that 1) thirty five microRNAs were differentially expressed in CTEPH patients, among which, a signature of 17 microRNAs, which was shown to be related to the disease pathogenesis by in silico analysis, gave diagnostic efficacy of both sensitivity and specificity >0.9. 2) Let-7b, one of the down-regulated anti-oncogenic microRNAs in the signature, was validated to decrease to about 0.25 fold in CTEPH patients. 3) ET-1 and TGFBR1 were direct targets of let-7b. Altering let-7b level influenced ET-1 and TGFBR1 expression in pulmonary arterial endothelial cells (PAECs) as well as the migration of PAECs and pulmonary arterial smooth muscle cells (PASMCs). These results suggested that CTEPH patients had aberrant microRNA signature which might provide some clue for pathogenesis study and biomarker screening. Reduced let-7b might be involved in the pathogenesis of CTEPH by affecting ET-1 expression and the function of PAECs and PASMCs.

Translational Advances in the Field of Pulmonary Hypertension.Translating MicroRNA Biology in Pulmonary Hypertension. It Will Take More Than “miR” Words

“…Other miRNAs with known causative actions in PAH, such as the miR-130/301 family (20) and miR-210 (14), have been found to be elevated in the pulmonary circulation of patients with PH. Moreover, differential expression of plasma miRNAs was also reported in acute pulmonary embolism (77) and chronic thromboembolic PH (78). Direct evidence has been presented regarding the transport and signaling of miR-143 among PAECs and PASMCs in PAH (71).…”

microRNAs in cardiovascular disease – clinical application

“…More recently, anti-oncogenic miRNA Let-7b was identified to be down-regulated in plasma of CTEPH patients [48]. These results gave rise for the exploration of miRNAs in the diagnosis of acute pulmonary embolism (APE) -a diagnosis for which there is no specific biomarker.…”

Discerning functional hierarchies of microRNAs in pulmonary hypertension

“…Further identifying miRNAs with shared disease association may offer insights into the relationship of PH to the multitude of its associated secondary diseases or systemic complications beyond the vasculature (2). For example, a number of miRNAs, including let-7 family members (85,86), are particularly active in both acute pulmonary embolism and CTEPH (WHO Group 4 PH). Additionally, a polymorphism in the 3′ UTR in the fibrinogen-α gene (FGA) that affects the binding of miR-759 has been associated with CTEPH (87) and pulmonary embolism.…”

“…Other miRNAs have been reported as differentially expressed in the circulating plasma of PH patients, including miR-150 (106), miR-23a (107), miR-26a (108), and miR-125a (109), but their biology is less understood. Alteration of entire cohorts of plasma miRNAs has been reported in PH (107,110), as well as CTEPH (86). While these and other miRNAs are considered as prospective biomarkers in PH (Table 3), numerous pitfalls remain for their development as robust diagnostic tests, including issues with interindividual variability, reproducibility among patient cohorts, the tissue source of circulating RNAs, appropriate modality of testing, and the complexity of validating a true signature of miRNAs rather than a single biomarker.…”