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.

Toxoplasmosis is an important parasitic infection of man and animals. It is well-known that the progression and severity of disease depend on the immunological status of the host, but recent studies suggest that the genetics of the parasite can also play a role. Diagnosis based on clinical appearance and serology is not always easy. However, molecular methods do not depend on an immune response, and allow direct detection of the parasite in biological samples. Thus they can be used to establish a diagnosis when serological tests are not definitive. Multicopy sequences specific for Toxoplasma gondii, e.g., the B1 gene or the 529-bp sequence, are especially useful in molecular tests. Real-time PCR is very sensitive and is a promising technique that is capable of providing a quantitative result. Molecular methods are also used for genotypic characterisation of T. gondii isolates. Analysis of polymorphic sequences determines the precise strain. The choice of sequence is critical when undertaking studies on the correlation between clinical signs and symptoms of disease and the T. gondii genotype. Further studies involving direct genotyping of T. gondii from clinical samples are needed.

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.

No abstract

Thyroid hormone receptor beta (THRB) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of THRB in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of THRB in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in THRB and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, THRB CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. In silico analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting THRB transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3′UTR of THRB, and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous TRHB by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of THRB transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased THRB expression in ccRCC. In contrast, THRB is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of THRB in ccRCC tumors.