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A7775 - miR-487b Expression Modifies BMI-1 and SUZ12 in Fibroblasts and Revealed a Dual Role in Proliferation and Migration in Hypersensitivity Pneumonitis
Author Block: J. Cisneros1, M. Espina2, B. Ortiz1, F. Toscano2, A. Garcia2, I. Herrera1; 1Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Ciudad de México, Mexico, 2Laboratorio Nacional de Soluciones Biomiméticas para Diagnostico y Terapia, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
Hypersensitivity pneumonitis (HP) is a complex pulmonary disease produced by an exaggerated immune and inflammatory response to the inhalation of environmental antigens that can produce the activation and proliferation of fibroblasts. The pathogenic mechanism involved in the chronic/fibrotic phase of HP is not fully understood and there are no studies focused on the role of microRNAs in fibroblast from HP. Here we analyzed the expression of miR-487b in normal and HP fibroblasts and the effects of its inhibition or overexpression on migration and proliferation. miR-487b has been considered a tumor suppressor but its role in pulmonary fibroblasts remains without study. Methods Primary fibroblasts derived from normal human lung and from HP were cultured in complete medium and the expression of miR-487b was quantified by qPCR. In normal fibroblasts, miR-487 was inhibited with a microRNA hairpin whereas in fibroblasts from patients, overexpression was achieved with a double-stranded RNA (microRNA mimic). Before and after transfection, expression of BMI-1 and SUZ12 was examined by western blot. Migration was evaluated by wound healing assay, proliferation was measured with WST and CyQuant. Apoptosis was determined by annexin-V staining assessed by flow cytometry. Results First, we quantified miR-487b expression in fibroblasts from normal (n=6), HP (n=7) and idiopathic pulmonary fibrosis (IPF, n=6), another interstitial lung disease, and found that miR-487b is significantly downregulated in HP and IPF fibroblasts. There were not differences between HP and IPF fibroblasts. Fibroblasts from HP patients showed increased migration and proliferation, and the expression of two targets of miR-487b, BMI-1 and SUZ12, was also increased when compared to normal fibroblasts. In these normal fibroblasts, inhibition of miR-487b causes upregulation of BMI-1 and SUZ12, reduces proliferation and has not significant effect on migration. On the other hand, the gain of function with an RNA mimic in HP fibroblasts provokes a decrease in BMI-1 and SUZ12 proteins and reduces the proliferative profile; however, the gain of miR-487b produces an increase of migration. Additionally, we found an increase of apoptosis in normal cells versus HP fibroblasts. Conclusions Our findings suggest that miR-487b might play a role in the proliferation of fibroblasts from patients with hypersensitivity pneumonitis by reducing progression towards a fibrotic phenotype.