Thyroid hormone receptor β1 (TRβ1) is a hormone-dependent transcription factor activated by 3,5,3'-l-triiodothyronine (T3). TRβ1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRβ1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRβ1 5' and 3'UTRs on protein translation. We determined TRβ1 expression and alternative splicing of TRβ1 5' and 3'UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1, a TRβ1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRβ1 3'UTR) were also determined. TRβ1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRβ1 UTR variants indicated that reduced TRβ1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5'UTRs and miRNA-204. The tumor suppressor activity of TRβ1 indicates that reduced TRβ1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.

Thyroid hormone (T(3)) regulates proliferation and differentiation of cells, via its nuclear receptors (TRs). These processes have been shown to be abnormally regulated during carcinogenesis. We have previously found aberrant expression of TRalpha and TRbeta mRNAs in renal clear cell carcinoma (RCCC), suggesting possible involvement of TRs in the carcinogenesis of RCCC. To understand the molecular actions of TRs in RCCC, cDNAs for TRbeta1 and TRalpha1 were cloned from 22 RCCC tissues and 20 surrounding normal tissues. Mutations were found in seven TRbeta1 and three TRalpha1 cDNAs. Two TRbeta1 cDNAs had a single mutation, while five TRbeta1 and three TRalpha1 had two or three mutations. Most of the mutations were localized in the hormone-binding domain. Using the TRs prepared by in vitro transcription/translation, we found that these mutations led to a loss of T(3) binding activity and/or impairment in binding to thyroid hormone response elements (TREs). Furthermore, nuclear extracts from RCCC tissues also exhibited impairment in binding to TREs. These results indicate that the normal functions of TRs in RCCC tissues were impaired. Together with the aberrant expression patterns, these mutated TRs could contribute to the carcinogenesis of RCCC.

MiRs up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.

Type 1 iodothyronine deiodinase (DIO1) catalyses the conversion of prohormone thyroxine to the active thyroid hormone 3,3′,5-triiodothyronine (T3), important regulator of cell proliferation and differentiation. DIO1 expression is reduced in the most common type of kidney neoplasia, clear cell Renal Cell Carcinoma (ccRCC). MicroRNAs are small, non-coding RNAs that regulate gene expression at posttranscriptional levels. The aim of this study was to analyze the potential regulation of DIO1 expression by microRNAs in ccRCC. Bioinformatic analysis revealed that 3′UTR of the human DIO1 gene transcript contains miR-224 and miR-383 target sites, which are conserved across mammalian species. Semi-quantitative real-time PCR was used to analyze the expression of miR-224 and miR-383 in 32 samples of ccRCC tumors (T) and in 32 matched control (C) samples. We observed statistically significant (p = 0.0002) more than four fold increase in miR-224 expression and nearly two fold increase in miR-383 expression in samples T compared to samples C. Tumor specific changes in expression of miR-224 negatively correlated with changes in DIO1 expression and intracellular T3 concentration. Transfection of HeLa cell line with miR-224 and miR-383 suppressed the activity of a luciferase reporter containing the 3′UTR of DIO1. This was abolished when constructs mutated at the miR-224 and miR-383 target sites were used instead, indicating that miR-224 and miR-383 directly bind to DIO1 3′UTR. Finally, induced expression of miR-224 in Caki-2 cells resulted in significant (p<0.01) reduction of DIO1 mRNA. This study provides a novel miRNA-mediated regulatory mechanism of DIO1 expression in ccRCC.

Summary. A reliable method has been developed for the determination of total serum T3, dialyzable fraction (DFT3), and absolute concentration of free T3 (AFT3). Total T3 values (mean ± SD) were: healthy euthyroid subjects, 0.33 ± 0.07 /Ag per 100 ml; hyperthyroid patients, 0.71 ± 0.1 jug per 100 ml; hypothyroid, 0.10 + 0.03 jug per 100 ml. Values (mean ± SD) for DFT3 in these groups were 0.46 + 0.14%, 0.78 ± 0.17%, and 0.16 ± 0.08%, respectively. Calculated values for AFT3 were: 1.51 + 0.4 m/tg per 100 ml, 5.00 ± 0.6 mug per 100 ml and 0.24 ± 0.1 mug per 100 ml, respectively. Dilution of serum before dialysis lowered estimated DFT3 values. Enrichment of serum with labeled T3 in the range examined did not affect DFT3. However, DFT3 was increased by addition of Merthiolate to serum in concentration 1: 10,000 due to displacement of T3 from thyroxine-binding globulin to albumin. The data suggest that triiodothyronine may play a considerably more important role in normal and pathological physiology, as evidenced by kinetic analysis using these data. A metabolic role for T3 equal to that of T4 is indicated.