The role of miR-139-5p in radioiodine-resistant thyroid cancer

Abstract

Purpose: Radioiodine I-131 (RAI) is the therapy of choice for differentiated thyroid cancer (DTC). Between 5% and 15% of DTC patients become RAI refractory, due to the loss of expression/function of iodide metabolism components, especially the Na/I symporter (NIS). We searched for a miRNA profile associated with RAI-refractory DTC to identify novel biomarkers that could be potential targets for redifferentiation therapy.

Methods: We analyzed the expression of 754 miRNAs in 26 DTC tissues: 12 responsive (R) and 14 non-responsive (NR) to RAI therapy. We identified 15 dysregulated miRNAs: 14 were upregulated, while only one (miR-139-5p) was downregulated in NR vs. R tumors. We investigated the role of miR-139-5p in iodine uptake metabolism. We overexpressed miR-139-5p in two primary and five immortalized thyroid cancer cell lines, and we analyzed the transcript and protein levels of NIS and its activation through iodine uptake assay and subcellular protein localization.

Results: The finding of higher intracellular iodine levels and increased cell membrane protein localization in miR-139-5p overexpressing cells supports the role of this miRNA in the regulation of NIS function.

Conclusions: Our study provides evidence of miR-139-5p involvement in iodine uptake metabolism and suggests its possible role as a therapeutic target in restoring iodine uptake in RAI-refractory DTC.

Keywords: Na/I symporter (NIS); Radioiodine resistance; Thyroid cancer; miR-139-5p.

Fig. 1

miRNA expression levels in non-responder and responder patients. A Study design and heat map and hierarchical clustering of 24 miRNAs differentially expressed in DTC tissue biopsies from non-responder (NR) and responder (R) patients. The expression levels of each miRNA were expressed as ΔCt value normalized using U6 as endogenous control. The expression level of each miRNA is indicated by a color scale (red for high expression, green for low expression). B 15 miRNAs most significantly dysregulated in DTC tumor tissues from NR and R subsets. Relative expression levels of each miRNA are reported as mean ± SD normalized to the endogenous control. C Validation of miR-139-5p and miR-21-5p expression levels in NR and R patients using real-time PCR analysis. Expression levels of miR-139-5p are significantly lower in NR patients than in R patients (1 ± 1.179 vs. 0.4372 ± 0.3958); those of miR-21-5p were significantly higher in NR patients than in R patients (1 ± 1.043 vs. 1.866 vs. 1.657). Data are expressed as 2.^−ΔΔCt value (mean ± SD), normalized to the endogenous control (snRNA U6). P values were obtained using Mann–Whitney U test; *p value < 0.05

Fig. 2

miR-139-5p relative expression levels in thyroid cancer cell lines. Expression levels of miR-139-5p in A primary cell lines, and B immortalized cell lines. Data are expressed as 2-ΔΔCt value (mean ± SD), normalized to the endogenous control (snRNA U6), and compared with primary normal cell lines A or a commercial pool of normal thyroid tissues (B) (grey bars), p value < 0.05, *; 0.005 **; 0.0005 *** (t test data)

Fig. 3

Thyroid-specific gene expression in primary cell lines. Expression levels of thyroid-specific genes (NIS, TPO, TSHR, TG, and PAX8) in primary cell lines 48 h after miR-139-5p overexpression. Data are expressed as 2-ΔΔCt value (mean ± SD), normalized to the endogenous control (GAPDH), and compared with control (CTRL) samples, (cells transfected with the negative control), arbitrarily set at 1, p value < 0.05, *; 0.005 **; 0.0005 *** (t test data)

Fig. 4

Thyroid-specific gene expression in immortalized cell lines. Expression levels of thyroid-specific genes (NIS, TPO, TSHR, TG, and PAX8) in immortalized cell lines after 48 h from miR-139-5p overexpression. Data are expressed as 2-ΔΔCt value (mean ± SD), normalized to the endogenous control (GAPDH), and compared with control (CTRL) samples (cells transfected with the negative control), arbitrarily set at 1, p value < 0.05, *; 0.005 **; 0.0005 *** (t test data)

Fig. 5

Intracellular iodine levels. Intracellular iodine in primary tumor cell line 1, line 2, and immortalized cell lines (TPC1, BCPAP, K1, 8505C, and SW1736) treated or not with KClO4 10 µM. The quantity of iodine is expressed as absolute concentration (nM) in cells with overexpressed miR-139-5p (miR-139-5p) as compared with control (CTRL) cells. p value < 0.05, *; 0.005 **; 0.0005 *** (t test data in technical replicates)

Fig. 6

NIS subcellular localization in primary cell lines. NIS-stained (green) in primary cell line 2 normal cells, and primary cell line 2 tumor cells. Immunofluorescence was performed before (CTRL) and after miR-139-5p overexpression (o.e. miR-139-5p). Images are reported at 20X magnification, and nuclei were Hoechst-stained (blue)

Fig. 7

NIS subcellular localization in immortalized cell lines. Left panel: NIS-stained (green) TPC1 A, BCPAP B, K1 C, 8505c D, and SW1736 E cells before (CTRL) and after miR-139-5p overexpression (o.e. miR-139-5p). Images are reported at 40X magnification. Nuclei were Hoechst-stained (blue). White arrows indicate the membrane of the cells. Central panel: representative western blot (one out of the three biological replicates) of NIS (90 KDa) levels in nuclear, cytoplasmic, and membrane fractions from each line in CTRL and o.e. miR-139-5p samples. The fraction loading controls are represented by the levels of H3 (15 KDa) for nuclear fractions, actin for the cytoplasm (42 KDa) and LDLR (95 KDa) for membranes. The loading control of the total extract from each cell line is reported in Supplementary Fig. 3. Right panel: NIS relative levels from densitometric analysis of three biological replicates; statistical analysis was performed comparing the membrane fraction of o.e. miR-139-5p with CTRL cells. p value < 0.05, * (t test data). The uncropped membranes are reported in Supplementary File 1 as high-resolution images