SAT-549 Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Primary Human Thyrocytes

Abstract

Thyroid gland, as one crucial endocrine organ to synthesize and secrete thyroid hormones directly response to TSH, is indispensable to human health. The secretion of exosomes by FRTL-5 normal thyroid cell line and TPC-1 papillary thyroid cancer cell line has been verified. However, the verification and profiling of exosomes secreted by human thyroid and a possible role for exosomes in thyroid function has not been explored. To investigate whether human thyroid could secrete exosomes, normal thyroid tissue from the surgical thyroidectomy of benign follicular nodules were collected and digested. Primary thyrocytes were cultured with or without TSH, and the medium was collected. Exosomes were isolated via differential ultracentrifugation, observed under transmission electron microscopy, measured by nanoparticle tracking analysis, and confirmed by immunoblot. Further, label-free liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was conducted to investigate the proteomic profiling of thyrocytes exosomes. In the present study, secreted exosomes were verified in primary human thyrocytes. Exosomes exhibited the same shape and size (102.80±36.01 vs. 95.82 ±30.10 nm, p>0.05) as control exosomes after TSH stimulation, but importantly, the total number of exosomes increased significantly after TSH stimulation for 48 h (3.46±0.56×107 vs. 1.98 ±0.20 ×107 nm, p<0.05). The mRNA level of Rab27b, the protein crucial for late endosomal formation, was also increased significantly after TSH stimulation, indicating that TSH activated the biogenesis of exosomes. Overall, 202 unique proteins were identified in thyrocytes exosomes. These included multiple proteins known to be involved in the endosomal pathway, such as annexins, and several cytoskeletal, cytoskeletal binding-proteins and motor proteins. One significant identified protein was thyroglobulin. Among the identified proteins, 77 proteins exhibited a differential expression pattern in TSH exosomes compared with the control exosomes, which mainly participate in cellular process, metabolic process, biological regulation, response to stimulus and regulation of biological process. This study provides direct evidence that human thyroid secretes exosomes. The characterization and proteomic profiling of exosome secreted by primary human thyrocytes provides potential physiological significance of exosomes in thyroid function.