Identification of Exosomal microRNAs and Their Targets in Papillary Thyroid Cancer Cells

Abstract

The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA–target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs’ targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.

RNA Profile of Cell Bodies and Exosomes Released by Tumorigenic and Non-Tumorigenic Thyroid Cells

“…Several studies have analyzed the role of miRNAs circulating in extracellular vesicles as biomarkers in thyroid cancer [26][27][28][29]. Recently, a high expression of miR-31-5p, miR-222-3p and let-7i-3p was found in exosomes isolated from TPC-1 and K1, two thyroid cancer cell lines [13] and extracellular vesicles isolated from thyroid tissue after BRAFV600E activation [30]. Exosomes naturally released from distinct cell types can also have beneficial effects in several human diseases.…”

“…In a previous report, we published results related to the characterization of Nthy-ori 3.1 and TPC-1 cell-derived exosomes by Dynamic Light Scattering (DLS) analysis and Western blotting analysis [13]. From the DLS analysis, we found that exosomes had a size distribution in the expected range of 40-160 nm.…”

“…The media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA; code 10500064), penicillin (100 IU/mL), streptomycin (0.1 mg/mL) (Thermo Fisher Scientific Inc., Waltham, MA, USA; code 15140-122) and amphotericin B (2.5 µg/mL) (Thermo Fisher Scientific Inc., Waltham, MA, USA; code 30-003). The cells were grown in a monolayer at 37 • C in 5% CO 2 and humidified, as previously described [13]. To confirm the identity of the cell lines, short tandem repeat analysis was performed by using an AmpFLSTR NGM SElect PCR Amplification Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; code 4457889).…”

Role of Extracellular Vesicles in Thyroid Physiology and Diseases: Implications for Diagnosis and Treatment

“…Identifying EV-miRNA targets and studying miRNA-mRNA networks in a global and unbiased manner greatly benefited from the development of bioinformatics tools. A comparison of the profile of EV-miRNAs released by two thyroid cancer cell lines with that of non-cancerous thyroid cells revealed a differential abundance of five miRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) in EVs of tumor cells, with three of them (miR-31-5p, miR-222-3p, and let-7i-3p) being more abundant in EVs from the more aggressive cancer cells [ 41 ]. These miRNAs were also present in EVs isolated from thyroid tissue after BRAF V600E induction [ 22 ].…”

“…These miRNAs were also present in EVs isolated from thyroid tissue after BRAF V600E induction [ 22 ]. Functional enrichment and network analysis of these miRNAs’ target genes (ICAM1, FOXO3, SELE, ETS1, RECK, PTEN, and TIMP3) are regulators of these miRNAs from aggressive thyroid cancer cells of the tumor microenvironment through modulation of angiogenesis and immune response [ 41 ].…”