MicroRNA mapping of bronchial aspirate for molecular phenotyping and prognostication in patients on mechanical ventilation

Abstract

The application of microRNA (miRNA) profiling in respiratory biospecimens, particularly bronchial aspirate (BAS), remains underexplored. Here, we aimed to validate and refine miRNA quantification in BAS samples to establish its suitability for molecular phenotyping. This was a multicenter study including 288 COVID-19 patients on invasive mechanical ventilation. Respiratory biospecimens included BAS, tracheal aspirate, and bronchoalveolar lavage fluid samples. A predesigned miRNA panel was evaluated using RT-qPCR. Biomarker evaluation and functional assessment were subsequently conducted. An initial technical validation phase corroborated the reproducibility of miRNA profiling in BAS samples. Comparative analyses of miRNA expression profiles across respiratory samples revealed distinct miRNA patterns among biospecimens. In the biomarker analysis, two miRNA ratios, miR-34c-5p/miR-34a-5p and miR-34c-5p/miR-125b-5p, were inversely associated with intensive care unit (ICU) survival (hazard ratio [HR]: 0.18 and 0.17, respectively) during the discovery phase. Risk and survival analyses in the test phase confirmed the reproducibility of the miR-34c-5p/miR-34a-5p ratio (hazard ratio [HR] = 0.17). Functional analyses revealed the utility of miRNA profiling in BAS for identifying pathogenic pathways and developing therapeutic targets. Overall, these findings position miRNA profiling in BAS samples as a valuable approach for biomarker discovery, identification of pathophysiological mechanisms, and development of targeted pulmonary therapies.

This work was supported by the Instituto de Salud Carlos III de Madrid (COV20/00110), cofunded by the European Union and Centro de Investigación Biomedica En Red—En fermedades Respiratorias (CIBERES). CIBERES (CB07/06/2008) is an initiative of the Instituto de Salud Carlos III. Further support was provided by the Programa de ciones “estar preparados” UNESPA (Madrid, Spain). F.B. is supported by the ICREA Academia Program. D.d.G.-C. has received financial support from Instituto de Salud Carlos III (Miguel Servet 2020: CP20/00041), cofunded by the European Union. M.M. (PFIS 2021: FI21/00187) and M.P.-P. (PFIS 2023: FI23/00022) are the recipients of a doctoral fellowship from Instituto de Salud Carlos III and cofunded by the European Union. MCGH held a predoctoral fellowship from Ayudas al Personal Investigador en Formación from IRBLleida/Diputación de Lleida. C.R. is supported by Departament de Salut (Pla Estratègic de Recerca i Innovació en Salut (PERIS): SLT028/23/000191) and programa predoctoral AGAUR-FI ajuts (2025 FI-2 00863) Joan Oró de la Secretaria d’Universitats i Recerca del Departament de Recerca i Universitats de la Generalitat de Catalunya i del Fons Europeu Social Plus. We want to particularly acknowledge the patients and the Biobanco A Coruña—Área Sanitaria A Coruña y Cee—SERGAS—INIBIC (B.0000796, PT17/0015/0032) and IdISBa Biobank and CIBERES Pulmonary Biobank Consortium (PT17/0015/0001), both integrated in the Spanish National Biobanks Network financed by the Carlos III Health Institute and the Units of Intensive Care, Clinical Analysis and Pulmonology of Hospital Universitario Son Espases, and Hospital Son Llatzer for their collaboration. This work was supported by the IRBLleida Biobank (B.000682) and the Biobank and Biomodels Platform ISCIII PT23/00032. Human sample manipulation was performed at the Cell Culture Facility, Universitat de Lleida (Lleida, Catalonia, Spain).