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

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.

Role of phosphatidylinositol 3-kinase-protein kinase B signaling pathway in Na+-I-symporter expression for lactating breast cells / 中华地方病学杂志

Objective To elucidate the function of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway underlying the regulation of Na+-I-symporter (NIS) and the influence of different levels of iodine on PI3K-AKT signaling pathway in lactating breast cells.Methods The primary cultured mammary gland cells were divided into three groups:①control group [0 μmol/L LY294002 + 0 μg/L insulin-like growth factor Ⅰ (IGF-Ⅰ)];②stimulation group (50 μg/L IGF-Ⅰ);③inhibition group (40 μmo]/L LY294002 + 50 μg/L IGF-Ⅰ).In addition,the cells were treated with different iodine contents (0,5,50,1 000,3 000 μg/L) for low iodine groups 1 and 2,iodine group,high iodine groups 1 and 2,and IGF-Ⅰ (50 μg/L) was used to stimulate PI3K-AKT signaling pathway.The expressions of AKT and NIS mRNA and protein were determined by real-time quantitative PCR and Western blotting,respectively.Results The expression of AKT mRNA (1.497 ± 0.550) in stimulation group was higher than that in inhibition group (0.777 ± 0.108,P ＜ 0.05),while the expression of NIS mRNA and protein in stimulation group (0.783 ± 0.187,0.618 ± 0.103) was lower than those in inhibition group (2.430 ± 1.423,1.417 ± 0.250,all P ＜ 0.05).With the iodine concentration increasing,except high iodine group 1 (1.090 ± 0.356),the expression of AKT mRNA in low iodine groups 1 and 2,iodine group,high iodine group 2 (1.758 ± 0.893,1.320 ± 0.538,1.003 ± 0.006,0.745 ± 0.307) tended to decline;total AKT protein (0.640 ± 0.106,0.601 ± 0.081,0.583 ± 0.089,0.555 ± 0.097,0.532 ± 0.023) and NIS mRNA (2.259 ± 0.682,1.823 ± 0.332,1.409 ± 0.366,1.321 ± 0.405,1.150 ± 0.454) tended to decline in low iodine groups 1 and 2,iodine group,high iodine groups 1 and 2;except low iodine group 2 (0.484 ± 0.179),NIS protein expression tended to decline (0.556 ± 0.199,0.502 ± 0.179,0.455 ± 0.126,0.435 ± 0.138);however,except low iodine group 2 (0.076 ± 0.045),the p-AKT protein expressions (0.078 ± 0.049,0.079 ± 0.040,0.085 ± 0.055,0.095 ± 0.051) were on the rise.Conclusion PI3K-AKT signaling pathway may play an inhibition role in the expression of NIS in lactating breast cells.

Effects of BRAF(V600E) mutation on Na(+)/I(-) symporter expression in papillary thyroid carcinoma.

Radioiodine ablation (RIA) therapy is one of the most important treatments for papillary thyroid carcinoma (PTC), but some patients who received (131)I have radioiodine-refractory disease caused by the decreased expression of the Na(+)/I(-) symporter (NIS). BRAF(V600E) mutation is one possible risk factor that can disturb the NIS expression, but the roles are unclear in clinical practice. This research discussed the association of BRAF(V600E) mutation and NIS expression in PTC tissue and the clinical implications in RIA therapy. 134 PTC samples were collected between June 2013 and June 2014 from Tongji Hospital affiliated to Tongji Medical College, and their clinical characteristics were analyzed. RT-PCR was used to detect the BRAF(V600E) mutation from formalin-fixed paraffin-embedded samples, and immunohistochemistry was applied to detect the NIS expression. IPP software was used to calculate the relative expression quantity of NIS. We found that there was no significant correlation between the absorbance (A) values of NIS and clinicopathologic features in these cases, even thyroid stimulating hormone. BRAF(V600E) mutation showed inhibitory effect on the NIS expression without statistically significant difference in all PTC cases (ß=-0.0195, P=0.085), but in the subgroup without hashimoto's thyroiditis (HT), BRAF(V600E) mutation could significantly inhibit the NIS expression (ß=-0.0257, P=0.046). The results indicate that BRAF(V600E) mutation is correlated with a lower expression of NIS in PTCs without HT, suggesting the radioiodine-refractory effects during RIA therapy in these patients.

Effects of BRAF(V600E) mutation on Na(+)/I(-) symporter expression in papillary thyroid carcinoma / 华中科技大学学报（医学）（英德文版）

Radioiodine ablation (RIA) therapy is one of the most important treatments for papillary thyroid carcinoma (PTC), but some patients who received (131)I have radioiodine-refractory disease caused by the decreased expression of the Na(+)/I(-) symporter (NIS). BRAF(V600E) mutation is one possible risk factor that can disturb the NIS expression, but the roles are unclear in clinical practice. This research discussed the association of BRAF(V600E) mutation and NIS expression in PTC tissue and the clinical implications in RIA therapy. 134 PTC samples were collected between June 2013 and June 2014 from Tongji Hospital affiliated to Tongji Medical College, and their clinical characteristics were analyzed. RT-PCR was used to detect the BRAF(V600E) mutation from formalin-fixed paraffin-embedded samples, and immunohistochemistry was applied to detect the NIS expression. IPP software was used to calculate the relative expression quantity of NIS. We found that there was no significant correlation between the absorbance (A) values of NIS and clinicopathologic features in these cases, even thyroid stimulating hormone. BRAF(V600E) mutation showed inhibitory effect on the NIS expression without statistically significant difference in all PTC cases (β=-0.0195, P=0.085), but in the subgroup without hashimoto's thyroiditis (HT), BRAF(V600E) mutation could significantly inhibit the NIS expression (β=-0.0257, P=0.046). The results indicate that BRAF(V600E) mutation is correlated with a lower expression of NIS in PTCs without HT, suggesting the radioiodine-refractory effects during RIA therapy in these patients.

Assessment of the Effect of Two Distinct Restricted Iodine Diet Durations on Urinary Iodine Levels (Collected over 24 h or as a Single-Spot Urinary Sample) and Na(+)/I(-) Symporter Expression.

INTRODUCTION: A restricted iodine diet (RID) may be recommended for depletion of the whole-body iodine pool in patients with differentiated thyroid cancer referred for radioiodine treatment or a whole-body scan. Evaluation of the iodine pool is possible through urinary iodide (UI) measurements, which can be collected in 24-hour (24U) or spot urinary (sU) samples. However, the minimum period required for an RID to lower the iodine pool, the measurement of iodine in sU samples as a iodine pool marker, and the influence of the iodine pool on Na(+)/I(-) symporter (NIS) expression are debatable in the literature. OBJECTIVES: To compare the effects of 15- and 30-day RID on UI measurements in 24U and sU samples and the impact of RID on NIS expression. METHODS: Thyroidectomized patients went on a 15- or 30-day RID and collected 24U and sU samples before and after the RID. Twenty healthy individuals were evaluated for mRNA NIS expression before and after the RID. RESULTS: Of 306 patients, only 125 properly complied with both the RID and 24U collection. We observed a correlation between sU and 24U UI before the RID (n = 306, ρ = 0.47, p < 0.001), after a 15-day RID (n = 79, ρ = 0.49, p < 0.001), and after a 30-day RID (n = 46, ρ = 0.73, p < 0.001). There was a significant decrease in UI after the RID. The median UI measurement was 275 µg/l at baseline and 99 and 80 µg/l after a 15- and 30-day RID, respectively. There was a significant increase in NIS expression after a 15-day RID. CONCLUSIONS: A 15-day RID is sufficient to deplete the iodine pool. sU can replace 24U UI as a marker for assessing the iodine pool. NIS expression was increased after a 15-day RID.

The iodide-transport-defect-causing mutation R124H: a δ-amino group at position 124 is critical for maturation and trafficking of the Na+/I- symporter.

Na(+)/I(-) symporter (NIS)-mediated active accumulation of I(-) in thyrocytes is a key step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report novel findings derived from the thorough characterization of the ITD-causing mutation R124H, located in the second intracellular loop (IL-2). R124H NIS is incompletely glycosylated and colocalizes with endoplasmic reticulum (ER)-resident protein markers. As a result, R124H NIS is not targeted to the plasma membrane and therefore does not mediate any I(-) transport in transfected COS-7 cells. Strikingly, however, the mutant is intrinsically active, as revealed by its ability to mediate I(-) transport in membrane vesicles. Of all the amino acid substitutions we carried out at position 124 (K, D, E, A, W, N and Q), only Gln restored targeting of NIS to the plasma membrane and NIS activity, suggesting a key structural role for the δ-amino group of R124 in the transporter's maturation and cell surface targeting. Using our NIS homology model based on the structure of the Vibrio parahaemolyticus Na(+)/galactose symporter, we propose an interaction between the δ-amino group of either R or Q124 and the thiol group of C440, located in IL-6. We conclude that the interaction between IL-2 and IL-6 is critical for the local folding required for NIS maturation and plasma membrane trafficking.

Effects of High Levels Iodine Intake through Meal on mRNA Expression of Placental and Breast NIS in Rats / 环境与健康杂志

Objective To study the effects of excessive iodine intake through the meal on the expression of mRNA of placental NIS in pregnant rat and breast NIS in lactating rats. Methods Wistar rats, weaning one month, were randomly divided into three groups according to the body weights, i.e., normal iodine (NI), ten fold high iodine(10HI), one hundred fold high iodine(100HI), the ratio of female and male was 2∶1. Iodine intake of the groups were about 6.15, 61.5 and 615.0 mg/d respectively. After 3 months of treatment, the urine iodine was determined by As-Ce-catalytic spectrophotometry. The rats mated and had offspring. Their placenta and breasts were taken on the seventeenth day of pregnancy and tenth day of lactation respectively. Then NIS mRNA expression was determined by RT-PCR. Results The urine iodine increased with the increase of the iodine intake. The urine iodine and iodine intake showed the parallel magnification. Compared with the NI group,AR value of the placental NIS mRNA in 100HI group and the breast NIS mRNA in 10HI and 100HI group significantly decreased. Conclusion Excessive iodine intake may down-regulate the expression of the placental and breast NIS, which presents a protective effect on offspring.

Investigation of Value of Na~+/I~- Symporter Expression on Diagnosis of Thyroid Carcinoma and Prediction of ~(131)Ⅰ Therapeutic Effects / 中国普外基础与临床杂志

Objective To study the clinical value of Na+/I-symporter(NIS)expression on thyroid carcinoma diagnosis and 131I therapeutic effects prediction.Methods Thirty-one cases of thyroid carcinomas enrolled in this hospital from 1998 to 2006 were included.Using immunohistochemical method,NIS expression location,positive cell staining and expression intensity were observed,which was calculated by immunohistochemical scores(IHS)and NIS expression level was compared between primary and metastatic carcinoma.Results NIS was over-expressed on the basolateral membrane in positive control——Grave disease tissue,and showed no staining in negative control.NIS was expressed in cytoplasm in all 31 primary carcinomas,and IHS was over or equaled to 4 in 80.65% of them.Except for 2 no staining,NIS was expressed in cytoplasm in the rest 28 metastatic carcinomas.NIS expression was related to the pathological type of thyroid carcinoma,the strongest in PTC,then FTC,and the weakest in fvPTC.NIS expression in metastatic carcinoma was related to that in primary carcinoma.Conclusion NIS is over-expressed in cytoplasm in most thyroid carcinoma,and the iodide uptaking defect is mainly due to its wrong location.It has great potential to be applied in clinic by that it can help with the differential diagnosis of benign and malignant thyroid diseases,especially between FTA and FTC,and that it can help predict the therapeutic effects of 131I therapy following thyroid operation.

Relation Between Iodide Transporters and Thyroid Diseases / 中国普外基础与临床杂志

Objective To investigate the clinical significance of the three iodide transporters in thyroid diseases. Methods Literatures about the Na+/I-symporter (NIS), pendrin and human apical iodide transporter (hAIT) in recent years were reviewed and their expressions in different thyroid diseases were also analyzed. Results NIS proteins express at the basolateral membrane of thyrocytes in normal thyroid tissue, while pendrin and hAIT proteins are limited to the apical membrane of thyrocytes lining in the follicular lumen. In the tissues of thyroid carcinomas, it was found that the NIS proteins expressed in the cytoplasm and their expressions decreased; The mutation of NIS gene may be one of the main causes of congenital hypothyroidism. The expression of prendrin protein may be related to the function of follicles: its expression level increased significantly both in Graves diseases and toxic adenomas, but significantly decreased in differentiated thyroid carcinoma. However, the correlation between the decrease and the degrees of differentiation of carcinoma cell line are still disputable. The expression of hAIT protein does not significantly altered in hyperfunctioning tissues. It only slightly decreased occasionally in hypofunctioning adenomas, but it decreased significantly in thyroid carcinomas. Conclusion The abnormal expressions of the three iodide transporters may be related closely with the type of thyroid diseases. However, their pathogenic mechanisms and the causes of their abnormal expression are still unknown, which need to be studied further.