Abstract
Background and aims: Type 1 deiodinase (D1) catalyses synthesis of triiodothyronine (T3) which regulates the expression of many tumor suppressor genes and oncogenes. Splicing factors SF2/ASF and hnRNPA1 regulate alternative splicing which is often disturbed in cancers. The aim of the study was to analyze the expression of alternatively spliced transcripts of D1 in clear cell renal cell carcinoma (ccRCC), in correlation with expression of splicing factors.
Methods: Using semi-quantitative real-time PCR we analyzed: i) 19 tissue samples of ccRCC tumors (“T”); ii) 19 control samples (“C”) of the contralateral pole of cancer kidney not infiltrated by tumor; iii) 6 control samples (“N”) (nonneoplastic kidney abnormalities). The transcript variants of D1 were cloned from cDNA via AT-cloning in pGEM-T vector. Results: Cloning of D1 mRNA isoforms revealed different transcripts, consisting of combinations of 4, 3, 2 exons, and fragments of single exons. The expression of all transcript variants of D1 was dramatically (>90%) and statistically lowered in samples T compared to control samples C. The expression of SF2/ASF and hnRNPA1 mRNA in samples T was lowered of 63.5% +/- 8.8% S.D. and 55.5% +/- 10.8% S.D., respectively (p<0,05). The ratio of D1 mRNA isoforms encoding for protein variants possessing or lacking catalytically active center: a) was lowered in samples T of 68.8% +/- 12.9% S.D. (p<0,05); b) correlated with ratio of splicing factors SF2/ASF:hnRNPA1 in controls C but not in samples T. The ratio of splicing factors SF2/ASF:hnRNPA1 was lowered in samples T of 26.8% +/- 12.9% S.D. (p<0,05).
Conclusions: The alternative splicing of D1 mRNA is disturbed in ccRCC which possibly results from impaired expression of splicing factors SF2/ASF and hnRNPA1.