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.

Figure 1. Circulating miRNAs signature in CTEPH patients and healthy controls.

Heirarchical clustering and heatmap diagram of the optimum circulating miRNAs signature derived from the greedy search. RNA was isolated from EDTA-coagulated plasma from CTEPH patients (n = 10) and healthy controls (n = 10).

Figure 2. Independent validation of differentially expressed miRNAs.

Three candidate miRNAs (miR-602, let-7b and miR-22) from the microarray were validated by qRT-PCR in an independent cohort of CTEPH patients (n = 40) and healthy controls (n = 40). (A) Relative expression (2^-ΔCT) normalized to cel-miR-39 was shown as median with interquartile. P value was calculated by Mann-Whitney U test. ***P<0.001. (B) ROC curve analysis and AUC for 2 validated miRNAs (let-7b and miR-22) in the diagnosis of CTEPH.

Figure 3. ET-1 and TGFBR1 were direct target genes of let-7b.

EGFP reporter assay was applied for target validation in HEK 293 cells. Oligonucleotide mimics and negative control (NC) of let-7b were co-transfected with reporter plasmids and mutated controls, respectively. (A) & (B) Predicted duplex of let-7b and its target region of TGFBR1 and ET-1 with experimental mutation in seed sequences respectively. The number indicates the region position in its 3′-UTRs. The mutated nucleotide was shown in red, and the original nucleotide was its complementary one. (C) & (D) let-7b down-regulated EGFP expression through the predicted seed sequences in 3′-UTRs of ET-1 and TGFBR1. P value was calculated by two-sample Kolmogorov-Smirnov test. * P<0.05.

Figure 4. Elevated plasma ET-1 level in CTEPH patients and its correlation with let-7b.

(A) Plasma endothelin-1 level of CTEPH patients (n = 40) and healthy controls (n = 40) measured by ELISA. P value was calculated by Mann-Whitney U test. P<0.001. (B) Spearman correlation and scatter plot of plasma endothelin-1 and let-7b (n = 80). The X- and Y-axis were log10 transformed. The correlation coefficient and P value were shown.

Figure 5. Altering let-7b regulated ET-1 and TGFBR1 expression in PAECs.

Human pulmonary arterial endothelial cells (PAECs) were infected with a let-7b antagonist/\mimics lentivirus or empty control. (A) Endogenous let-7b was decreased to a level about 55% by antagonist lentivirus and was increased to about 2 fold by mimics. (B) After normalized by β-actin, TGFBR1 expression was increased about a half in let-7b antagonist infected PAECs and slightly decreased in let-7b over-expressed cells by western blotting. (C) Culture medium was collected after continuous culture for 48 h, and ET-1 levels were detected by ELISA. ET-1 level in culture medium of let-7b over-expressed PAECs was obviously decreased, and was increased in let-7b antagonized PAECs. (D) Let-7b antagonized cells were transfected with siRNA for TGFBR1 and control siRNA respectively. Silencing TGFBR1 with siRNA could reverse the increased ET-1 level causing by let-7b antagonist (n = 5). P value was calculated by two-sample Kolmogorov-Smirnov test. *** P<0.001. * P<0.05.

Figure 6. Let-7b regulated PAECs and PASMCs migration.

Human PAECs or PASMCs were transfected with a let-7b antagonist lentivirus or let-7b mimics, and the migration was evaluated by wound healing assays. Pictures were taken at 0 h, 12 h, 24 h, and 48 h. The wounded area was expressed as the percentage of recovery. (A) Let-7b mimics suppressed PAECs migration, and its antagonist promoted TGF-β induced PAECs migration at 24 h (n = 5). (B).Let-7b mimics suppressed PASMCs migration, and its antagonist promoted TGF-β induced PASMCs migration at 24 h (n = 5). P value was calculated by two-way ANOVA, and Post Hoc Test was done by Student-Newman-Keuls method. *** P<0.001. * P<0.05.