Overexpression of miR-3168 impairs angiogenesis Pulmonary Arterial Hypertension: Insights from circulating miRNA analysis.

Background Pulmonary Arterial Hypertension (PAH) is a rare disease where the thickening of the precapillary pulmonary arteries ends up inducing right heart failure. Nowadays, obtaining an early diagnosis is challenging and typically delayed until undergoing right-heart catheterization. Methods We performed small RNA sequencing (microRNA-seq) in the plasma of idiopathic PAH patients and controls, that we validated by qPCR. We then interrogated the role of miR-3168 in HUVECs by performing western-blot, flow cytometry and tube formation assays. Results We found 29 differentially expressed microRNAs and validate 7 of them let-(7a-5p, let-7b-5p, let-7c-5p, let-7f-5p, miR-9-5p, miR-31-5p, miR-3168) in a nationwide cohort of 120 patients and 110 controls. We then used classification models to analyze their potential as PAH predictor. In the first half of our cohort, we obtained a model with an AUC of 0.888. Although, this value lowered to 0.738 after using this model in the whole cohort of patients. Additionally, we validated the effect of miR-3168, a novel upregulated miRNA in PAH patients which targets BMPR2, and impairs angiogenesis, as assessed by the tube formation assay. Conclusion We identified novel downregulated and upregulated microRNAs in idiopathic PAH patients, developed a 3-microRNA signature for diagnosis, and validated in vitro that miR-3168 targets BMPR2, thereby impairing angiogenesis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was partially funded by grants from Fundacion Contra la Hipertension Pulmonar, Jansen Pharmaceuticals, Ministerio de Ciencia e Innovacion (PI18/01233, Xunta de Galicia Axudas para consolidacion e estructuracion de unidades de investigacion competitivas e outras accions de fomento (GRC-ED431C 2022/26) to DV. MLD was supported by a Xunta de Galicia predoctoral fellowship (ED481A-2018/304). AIG was supported by a collaboration grant from Ministerio de Educacion y Formacion Profesional 2020-21. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was conducted after the approval of the Comite de etica da Investigacion de Galicia (CEIC Galicia). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data generated in this article is available upon request. RNA-seq derived data is freely available at GSE222022, count matrix and DESeq2 analysis tables can be accessed in the supplementary dataset.