Abstract
Background and Objectives: Thyroid hormone(T3) is required for development and growth and essential for metabolic homeostasis. T3 acts via the nuclear receptors (TRalpha and TRbeta) to regulate target genes that control cell proliferation, differentiation and function. Abnormal expression and mutation of the THRB1 gene encoding TRbeta have been implicated in human carcinogenesis. We previously showed that alternative splicing of 5’- and 3’- untranslated region (UTR) exons in human THRB1 is dependent on the tissue type and aberrant in ccRCC. The aim of this study was to investigate effects of THRB1 5’- and 3’-UTR alternative splicing on TRbeta-1 protein expression in ccRCC.
Methods: RealTime PCR technology, Rapid Translation System RTS and protein detection techniques were used.
Results: Bioinformatic analysis of secondary structures in TRbeta-1 5’-UTRs revealed that all alternatively spliced variants are strongly folded especially in exon 2a which is present in all published THRB1 mRNA sequences. Moreover, rates of in vitro protein synthesis from vector-based constructs containing 5’UTRs were correlated with folding free energies of these untranslated regions but not with RNA transcription level which suggests that protein synthesis is regulated by UTRs in a sequence dependent manner. Using RealTime PCR we showed that expression of rare TRbeta-1 variants together with altered levels of expression of TRbeta-1 binding miRNAs in ccRCC could be responsible for the poor correlation between mRNA and protein levels, thus providing compelling evidence for UTR-dependent regulation of TR protein synthesis in ccRCC.
Conclusions: In ccRCC the presence of multiple THRB1 transcripts with varying 5’-UTRs and altered levels of TRbeta-1 binding miRNAs suggests a mechanism to account for aberrant TRbeta-1 protein expression and further implicates THRB1 in renal carcinogenesis.