A circulating MicroRNA signature for the diagnosis of pulmonary arterial hypertension and functional characterization of candidate miR-3168.

Pulmonary Arterial Hypertension (PAH) is a rare, progressive disorder characterized by pathological vascular remodeling of the pulmonary arteries, ultimately leading to right heart failure. Early diagnosis remains challenging, as clinical manifestations are often nonspecific, and definitive confirmation still requires invasive right heart catheterization. Circulating microRNAs (miRNAs) have emerged as promising biomarkers for cardiovascular diseases due to their plasma stability and direct involvement in disease pathophysiology. We performed small RNA sequencing on plasma samples from 25 IPAH patients and 10 healthy controls to identify differentially expressed miRNAs. Seven candidate miRNAs were selected for further validation by quantitative PCR in a multicenter cohort of 110 IPAH patients and controls. Logistic regression models were built to evaluate diagnostic performance. Functional studies for miR-3168 were performed using western blot and flow cytometry in Pulmonary Artery Endothelial Cells (PAECs), and tube formation assay in human umbilical vein endothelial cells (HUVECs). Our initial screen identified 29 differentially expressed miRNAs. Seven of these candidates, including members of the let-7 family and miR-3168, were successfully validated in the larger cohort. A diagnostic panel incorporating three miRNAs (let-7a-5p, miR-9-5p, and miR-31-5p) was developed, which achieved an area under the curve (AUC) of 0.862 (95% CI = 0.7481-1) for discriminating PAH patients from controls. Separately, we investigated the functional role of miR-3168, which was upregulated in PAH patients. In PAECs, overexpression of miR-3168 led to a reduction of BMPR2 protein levels. Moreover, miR-3168 overexpression impaired tube formation in HUVECs. Our study identifies a plasma-derived three-miRNA signature with strong potential for the non-invasive diagnosis of PAH. Furthermore, we implicate miR-3168 as a potential contributor to endothelial dysfunction through its regulation of BMPR2 and its anti-angiogenic effects in vitro. These findings reinforce the dual utility of circulating miRNAs as both clinically relevant non-invasive biomarkers and as tools to discover novel disease biology.