甲状腺癌核内包涵体的新认识：与自噬和 BRAFV600E 突变相关。
New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.
机构信息
Institute of Pathology, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.
Institute of Anatomy and Electron Microscopy Unit of Imaging Center Essen, University Hospital of Essen, University of Duisburg-Essen, Essen, Germany.
出版信息
PLoS One. 2019 Dec 16;14(12):e0226199. doi: 10.1371/journal.pone.0226199. eCollection 2019.
BACKGROUND
Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function.
METHODS
NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing.
RESULTS
In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins.
CONCLUSIONS
NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.
背景
核内包涵体（NI）在正常和肿瘤组织中已为人所知多年，是甲状腺乳头状癌（PTC）的诊断标准之一。BRAF 激活参与自噬等过程。肝细胞癌中的 NI 含有与自噬相关的蛋白质。我们的目的是阐明甲状腺癌（TC）中的 NI 是否具有生物学功能。
方法
分析了 107 例石蜡包埋的 TC 标本中的 NI，包括所有主要亚型。如果 NI 被染色完整的核膜标志物核纤层蛋白 A/C（lamin AC）完整地包围并完全封闭，则认为该 NI 为阳性。进行透射电子显微镜（TEM）、免疫组织化学（IHC）、免疫荧光（IF）和 3D 重建，以研究 NI 的内容和形状；通过下一代测序分析 BRAFV600E 突变。
结果
在 29%的 TC 中至少检测到一个 lamin AC 阳性的核内包涵体；在 PTC 中最为常见（76%）。TEM 分析显示，这些 NI 内存在变性的细胞器和异噬溶酶体；IF 染色核的 3D 重建证实了 NI 完全被核膜封闭，与细胞质没有任何接触。在 14-29%的病例中，NI 对自噬相关蛋白 LC3B、泛素、组织蛋白酶 D、p62/自噬体 1 和组织蛋白酶 B 呈阳性染色。双免疫荧光显示，LC3B 和泛素、p62 和泛素以及 LC3B 和 p62 在同一 NI 中存在共定位。仅在 PTC 中检测到的 BRAFV600E 突变与 NI/PTC 的数量显著相关（p = 0.042），与 NI 中自噬相关蛋白的免疫反应性显著相关（p≤0.035）。BRAF-IHC 显示，这些 BRAF 阳性甲状腺细胞中的一些细胞在 NI 中含有与自噬相关蛋白共定位的突变型 BRAF。
结论
TC 中的 NI 完全被核膜包围。NI 内存在自噬相关蛋白以及变性的细胞器和溶酶体蛋白酶提示它们参与自噬和蛋白水解过程。NI 中 BRAFV600E 蛋白是否以及如何降解需要进一步研究。
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