Department of Biochemistry and Molecular Biology, The Medical Centre of Postgraduate Education, Warsaw, Poland.
PLoS One. 2011;6(9):e24541. doi: 10.1371/journal.pone.0024541. Epub 2011 Sep 2.
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.
1 型碘甲状腺原氨酸脱碘酶 (DIO1) 催化前激素甲状腺素转化为活性甲状腺激素 3,3',5-三碘甲状腺原氨酸 (T3)，这是细胞增殖和分化的重要调节剂。DIO1 的表达在最常见的肾癌类型，透明细胞肾细胞癌 (ccRCC) 中降低。微小 RNA 是一种小的非编码 RNA，可在转录后水平调节基因表达。本研究旨在分析微小 RNA 对 ccRCC 中 DIO1 表达的潜在调节作用。生物信息学分析表明，人 DIO1 基因转录本的 3'UTR 包含 miR-224 和 miR-383 的靶位点，这些靶位点在哺乳动物物种中是保守的。半定量实时 PCR 用于分析 32 个 ccRCC 肿瘤 (T) 样本和 32 个匹配对照 (C) 样本中 miR-224 和 miR-383 的表达。我们观察到 T 样本中 miR-224 的表达显著增加了 4 倍以上（p=0.0002），miR-383 的表达增加了近 2 倍。与 C 样本相比，T 样本中 miR-224 的表达变化与 DIO1 表达和细胞内 T3 浓度的变化呈负相关。HeLa 细胞系中转染 miR-224 和 miR-383 抑制了含有 DIO1 3'UTR 的荧光素酶报告基因的活性。当使用突变构建体替代 miR-224 和 miR-383 的靶位点时，这种抑制作用被消除，表明 miR-224 和 miR-383 直接结合到 DIO1 3'UTR。最后，在 Caki-2 细胞中诱导 miR-224 的表达导致 DIO1 mRNA 显著减少（p<0.01）。本研究提供了 ccRCC 中 DIO1 表达的新型微小 RNA 介导的调节机制。
