Epigenetic regulation of thyroid hormone receptor beta in renal cancer.
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.
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland; Genomic Medicine, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland.
Anna
A
Wojcicka
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Agnieszka
A
Piekielko-Witkowska
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Hanna
H
Kedzierska
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Beata
B
Rybicka
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Piotr
P
Poplawski
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Joanna
J
Boguslawska
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Adam
A
Master
Department of Biochemistry and Molecular Biology, Centre of Postgraduate Medical Education, Warsaw, Poland.
Alicja
A
Nauman
D020413:3' Untranslated Regions / Q000235:genetics; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics; D037042:Thyroid Hormone Receptors beta / Q000235:genetics*
D020413:3' Untranslated Regions; C524942:MIRN155 microRNA, human; C555677:MIRN425 microRNA, human; D035683:MicroRNAs; D037042:Thyroid Hormone Receptors beta; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human