Espadinha Carla, Cavaco Branca Maria, Leite Valeriano
Molecular Endocrinology Group, Molecular Pathobiology Research Centre (CIPM), Portuguese Institute of Oncology of Lisbon Francisco Gentil, E.P.E., Lisbon, Portugal.
Thyroid. 2007 Jun;17(6):497-509. doi: 10.1089/thy.2006.0263.
Paired box gene 8/peroxisome proliferator-activated receptor gamma (PAX8PPARgamma) translocation is a molecular event associated with follicular thyroid tumorigenesis and is generated by a chromosomal rearrangement between PAX8 and PPARgamma genes. In this study, we investigated the effects of PAX8PPARgamma fusion protein on cell growth and on thyroid-specific gene expression in immortalized human thyroid cells (Nthy-ori 3-1).
PAX8PPARgamma-, PAX8-, and thyroid transcription factor-1 (TTF-1)-transfected cell culture models; count of live and dead cells; mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR; and protein analysis by western blotting and gel shift assays.
Cells transfected with the PAX8PPARgamma fusion gene showed higher cell viability at 24, 48, and 72 hours after transfection than cells transfected with control vectors. A PAX8 expression vector increased thyroglobulin (Tg), sodium/iodide symporter (NIS), and thyroid-stimulating hormone (thyrotropin) receptor (TSHR) mRNA levels in a dose-dependent manner. TTF-1 expression vector promoted a significant increase of Tg mRNA level, but had no effect on NIS and TSHR mRNA levels. PAX8PPARgamma transfectants presented a significant decrease in TSHR mRNA level compared to empty vector, but had no effect on Tg and NIS mRNA levels. PAX8 plus PAX8PPARgamma significantly lowered Tg and TSHR mRNA expression levels, but upregulated NIS mRNA level, compared to PAX8 plus control vector.
The results obtained with this in vitro system demonstrated that PAX8PPARgamma increases thyroid cell viability and has opposite effects on thyroid-specific gene expression, suggesting that the presence of this rearrangement may contribute to the malignant transformation of thyroid follicular cells.
配对盒基因8/过氧化物酶体增殖物激活受体γ（PAX8/PPARγ）易位是一种与甲状腺滤泡肿瘤发生相关的分子事件，由PAX8基因和PPARγ基因之间的染色体重排产生。在本研究中，我们研究了PAX8/PPARγ融合蛋白对永生化人甲状腺细胞（Nthy-ori 3-1）细胞生长及甲状腺特异性基因表达的影响。
构建PAX8/PPARγ、PAX8及甲状腺转录因子-1（TTF-1）转染的细胞培养模型；进行活细胞和死细胞计数；通过逆转录-聚合酶链反应（RT-PCR）和定量RT-PCR进行mRNA分析；通过蛋白质印迹法和凝胶迁移试验进行蛋白质分析。
转染PAX8/PPARγ融合基因的细胞在转染后24、48和72小时显示出比转染对照载体的细胞更高的细胞活力。PAX8表达载体以剂量依赖方式增加甲状腺球蛋白（Tg）、钠/碘同向转运体（NIS）和促甲状腺激素（TSH）受体（TSHR）的mRNA水平。TTF-1表达载体促使Tg mRNA水平显著增加，但对NIS和TSHR mRNA水平无影响。与空载体相比，PAX8/PPARγ转染细胞的TSHR mRNA水平显著降低，但对Tg和NIS mRNA水平无影响。与PAX8加对照载体相比，PAX8加PAX8/PPARγ显著降低Tg和TSHR mRNA表达水平，但上调NIS mRNA水平。
该体外系统获得的结果表明，PAX8/PPARγ可增加甲状腺细胞活力，并对甲状腺特异性基因表达产生相反影响，提示这种重排的存在可能有助于甲状腺滤泡细胞的恶性转化。
