Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53792, USA.
Endocr Pathol. 2019 Dec;30(4):262-269. doi: 10.1007/s12022-019-09589-y.
Long non-coding RNAs (lncRNAs) participate in transcription and in epigenetic or post-transcriptional regulation of gene expression. They also have roles in epithelial to mesenchymal transition and in carcinogenesis. Because lncRNAs may also have a role in thyroid cancer progression, we examined a group of thyroid tumors which included papillary thyroid carcinomas and anaplastic thyroid carcinomas to determine the specific lncRNAs that were upregulated during thyroid tumor progression. An RT Profiler PCR Array Human Cancer Pathway Finder consisting of 84 lncRNAs (Qiagen) and fresh tissues of normal thyroid, PTCs, and ATCs with gene expression profiling was used to determine genes upregulated and downregulated in ATCs. Two of the most highly upregulated genes, prostate cancer antigen 3 (PCA3) and HOX antisense intergenic RNA myeloid 1 (HOTAIRM1 or HAM-1), were selected for further studies using a thyroid tissue microarray(TMA) with formalin-fixed paraffin-embedded tissues of normal thyroid (NT, n = 10), nodular goiters (NG, n = 10), follicular adenoma (FA, n = 32), follicular carcinoma (FCA, n = 28), papillary thyroid carcinoma (PTC, n = 28), follicular variant of papillary thyroid carcinoma (FVPTC, n = 28), and anaplastic thyroid carcinoma (ATC, n = 10). TMA sections were analyzed by in situ hybridization (ISH) using RNAscope technology. The results of ISH analyses were imaged with Vectra imaging technology and quantified with Nuance® and inForm® software. The TMA analysis was validated by qRT-PCR using FFPE tissues for RNA preparation. Cultured thyroid carcinoma cell lines (n = 7) were also used to analyze for lncRNAs by qRT-PCR. The results showed 11 lncRNAs upregulated and 7 downregulated lncRNAs more than twofold in the ATCS compared with PTCs. Two of the upregulated lncRNAs, PCA3 and HAM-1, were analyzed on a thyroid carcinoma TMA. There was increased expression of both lncRNAs in ATCs and PTCs compared with NT after TMA analysis. qRT-PCR analyses showed increased expression of both lncRNAs in ATCs compared with NT and PTCs. Analyses of these lncRNAs from cultured thyroid carcinoma cell lines by qRT-PCR showed the highest levels of lncRNA expression in ATCs. TGF-β treatment of cultured PTC and ATC cells for 21 days led to increased expression of PCA3 lncRNA in both cell lines by day 14. These results show that the lncRNAs PCA3 and HAM-1 are upregulated during thyroid tumor development and progression and may function as oncogenes during tumor progression.
长链非编码 RNA（lncRNA）参与转录和基因表达的表观遗传或转录后调控。它们在上皮-间充质转化和致癌作用中也具有作用。由于 lncRNA 可能在甲状腺癌的进展中也具有作用，我们检查了一组甲状腺肿瘤，包括甲状腺乳头状癌和间变性甲状腺癌，以确定在甲状腺肿瘤进展过程中上调的特定 lncRNA。使用 RT Profiler PCR 阵列人类癌症途径查找器（Qiagen）和新鲜的正常甲状腺、PTC 和 ATC 组织的基因表达谱，确定 ATC 中上调和下调的基因。在使用福尔马林固定石蜡包埋组织的甲状腺组织微阵列（TMA）进行进一步研究时，选择了上调最明显的两个基因，前列腺癌抗原 3（PCA3）和 HOX 反义基因间 RNA 髓样 1（HOTAIRM1 或 HAM-1）正常甲状腺（NT，n=10）、结节性甲状腺肿（NG，n=10）、滤泡性腺瘤（FA，n=32）、滤泡状癌（FCA，n=28）、甲状腺乳头状癌（PTC，n=28）、滤泡状变体的甲状腺乳头状癌（FVPTC，n=28）和间变性甲状腺癌（ATC，n=10）。通过使用 RNAscope 技术的原位杂交（ISH）分析 TMA 切片。使用 Vectra 成像技术对ISH 分析的结果进行成像，并使用 Nuance®和 inForm®软件进行量化。使用用于 RNA 制备的 FFPE 组织通过 qRT-PCR 验证 TMA 分析。还使用 qRT-PCR 分析了培养的甲状腺癌细胞系（n=7）中的 lncRNA。结果表明，与 PTC 相比，ATC 中有 11 个 lncRNA 上调，7 个 lncRNA 下调超过两倍。上调的两个 lncRNA，PCA3 和 HAM-1，在甲状腺癌 TMA 上进行了分析。TMA 分析后，与 NT 相比，在 ATC 和 PTC 中均观察到这两种 lncRNA 的表达增加。qRT-PCR 分析显示，与 NT 和 PTC 相比，ATC 中这两种 lncRNA 的表达均增加。通过 qRT-PCR 分析培养的甲状腺癌细胞系中的这些 lncRNA，显示 ATC 中的 lncRNA 表达水平最高。用 TGF-β处理培养的 PTC 和 ATC 细胞 21 天后，第 14 天两种细胞系中的 PCA3 lncRNA 表达均增加。这些结果表明，lncRNA PCA3 和 HAM-1 在甲状腺肿瘤发生和发展过程中上调，并且在肿瘤进展过程中可能作为癌基因发挥作用。
