Genetic Unit, Department of Biology, University of Pisa, Pisa, Italy.
Humanitas Clinical and Research Centre-IRCCS, Milan, Italy.
Thyroid. 2021 Jul;31(7):1056-1066. doi: 10.1089/thy.2020.0366. Epub 2021 Mar 2.
Galectin-3 () is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, OR = 0.66; 95% confidence interval = 0.46-0.93; P = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, , , , and were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by -silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.
半乳糖凝集素-3() 是一种重要的糖蛋白，它在细胞外基质、细胞质和核内发挥作用，调节 TTF-1 和 TCF4 转录因子，参与甲状腺细胞的恶性转化。在 基因中，一个常见的单核苷酸多态性（SNP）（c.191C>A，p.Pro64His；rs4644），编码密码子 64 处脯氨酸到组氨酸的变异，已被广泛研究。然而，关于甲状腺癌中 rs4644 的数据尚缺乏。因此，本研究旨在评估 rs4644 SNP 作为分化型甲状腺癌（DTC）的风险因素，并确定其对甲状腺细胞转录组的影响。我们进行了一项病例对照关联研究，纳入了 1223 名对照和 1142 名连续无关的 DTC 患者，以评估 rs4644-P64H 与 DTC 风险之间的关联。我们使用非恶性细胞系 Nthy-Ori（rs4644-C/A）和 CRISPR/Cas9 技术，生成携带 rs4644-A/A 或 rs4644-C/C 纯合子的同源细胞系。然后，通过 RNA-seq 分析衍生和未修饰的亲本细胞的转录组。通过定量逆转录 PCR 验证差异表达的基因，并在基因沉默后进一步测试亲本 Nthy-Ori 细胞中的表达，以研究靶基因的表达是否依赖于半乳糖凝集素-3 水平。rs4644AA 基因型与 DTC 风险降低相关（与 CC 相比，OR=0.66；95%置信区间=0.46-0.93；P=0.02）。我们发现 rs4644 作为转录共调节剂影响半乳糖凝集素-3。在受 rs4644 影响的 34 个基因中，、、、和 特别有趣，因为它们的表达受 rs4644 影响（CC>AA 为第一个，AA>CC 为其他），在 Genotype-Tissue Expression（GTEx）生物库的 574 个甲状腺组织中也是如此。此外，这些基因的表达受 - 沉默调节。在 Nthy-Ori 细胞中使用邻近连接测定法，我们发现 TTF-1 的相互作用依赖于基因型。我们的数据表明，在甲状腺中，rs4644 是一个跨表达数量性状基因座，可通过调节 TTF-1 来改变下游基因的转录表达。
