Integrated miRNA and mRNA expression profiling identifies novel targets and pathological mechanisms in autoimmune thyroid diseases
Rebeca Martínez-Hernández,
Ana Serrano-Somavilla,
Ana Ramos-Leví,
Miguel Sampedro-Nuñez,
Alberto Lens-Pardo,
José Luis Muñoz De Nova,
Juan Carlos Triviño,
María Ujue González,
Lorena Torné,
Javier Casares-Arias,
Noa B. Martín-Cófreces,
Francisco Sánchez-Madrid,
Mónica Marazuela
Affiliations
Rebeca Martínez-Hernández
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
Ana Serrano-Somavilla
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
Ana Ramos-Leví
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
Miguel Sampedro-Nuñez
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
Alberto Lens-Pardo
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
José Luis Muñoz De Nova
Department of Surgery, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain
Juan Carlos Triviño
Sistemas Genómicos, Valencia, Spain
María Ujue González
Instituto de Micro y Nanotecnología, IMN-CNM, CSIC (CEI UAM+CSIC), Tres Cantos, Spain
Lorena Torné
Instituto de Micro y Nanotecnología, IMN-CNM, CSIC (CEI UAM+CSIC), Tres Cantos, Spain
Javier Casares-Arias
Department of Cell Biology and Immunology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científcas and Universidad Autónoma de Madrid, Madrid, Spain
Noa B. Martín-Cófreces
Department of Immunology, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa, Universidad Autónoma de Madrid, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Spain
Francisco Sánchez-Madrid
Department of Immunology, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa, Universidad Autónoma de Madrid, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Spain
Mónica Marazuela
Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Princesa, Universidad Autónoma de Madrid, C/ Diego de León 62, 28006 Madrid, Spain; Corresponding author.
Background: The mechanisms underlying autoimmune thyroid disease (AITD) remain elusive. Identification of such mechanisms would reveal novel and/or better therapeutic targets. Here, we use integrated analysis of miRNAs and mRNAs expression profiling to identify potential therapeutic targets involved in the mechanisms underlying AITD. Methods: miRNA and mRNA from twenty fresh-frozen thyroid tissues (15 from AITD patients and 5 from healthy controls) were subjected to next-generation sequencing. An anti-correlated method revealed potential pathways and disease targets, including proteins involved in the formation of primary cilia. Thus, we examined the distribution and length of primary cilia in thyroid tissues from AITD and controls using immunofluorescence and scanning electron microscopy, and parsed cilia formation in thyroid cell lines in response to inflammatory stimuli in the presence of miRNA mimics. Findings: We found that the expression of miR-21-5p, miR-146b-3p, miR-5571-3p and miR-6503-3p was anti-correlated with Enolase 4 (ENO4), in-turned planar cell polarity protein (INTU), kinesin family member 27 (KIF27), parkin co-regulated (PACRG) and serine/threonine kinase 36 (STK36) genes. Functional classification of these miRNA/mRNAs revealed that their differential expression was associated with cilia organization. We demonstrated that the number and length of primary cilia in thyroid tissues was significantly lower in AITD than in control (frequency of follicular ciliated cells in controls = 67.54% vs a mean of 22.74% and 21.61% in HT and GD respectively p = 0.0001, by one-way ANOVA test). In addition, pro-inflammatory cytokines (IFNγ and TNFα) and specific miRNA mimics for the newly identified target genes affected cilia appearance in thyroid cell lines. Interpretation: Integrated miRNA/gene expression analysis has identified abnormal ciliogenesis as a novel susceptibility pathway that is involved in the pathogenesis of AITD. These results reflect that ciliogenesis plays a relevant role in AITD, and opens research pathways to design therapeutic targets in AITD. Funding: Instituto de Salud Carlos III, Comunidad de Madrid, Grupo Español de Tumores Neuroendocrinos y Endocrinos, Ministerio de Economía y Empresa and FEDER. Keywords: microRNAs, mRNA, Next generation sequencing, Autoimmune thyroid disease, Graves’ disease, Hashimoto's thyroiditis, Cilia