Department of Thyroid Surgery, The First Hospital of China Medical University, Shenyang, 110001, Liaoning Province, China.
Endocrine. 2018 Mar;59(3):555-564. doi: 10.1007/s12020-017-1520-1. Epub 2018 Jan 11.
The long non-coding RNA GAS8 antisense RNA 1 (lncRNA GAS8-AS1) is a tumor suppressor in papillary thyroid cancer (PTC), but the mechanisms underlying how GAS8-AS1 regulates PTC biology remain unclear. Here, we evaluated the molecular function of GAS8-AS1 in regulating autophagy in PTC cell lines.
GAS8-AS1 was overexpressed and knocked down in PTC cell lines by transfecting with expression plasmids or short interfering RNAs (siRNAs). Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8). qRT-PCR and western blot were used to determine changes in expression of autophagy-related genes. Autophagy was evaluated by immunofluorescence and transmission electron microscopy.
Relative GAS8-AS1 expression was lower in the PTC cell lines, TPC1 and BCPAP, compared to a normal thyroid cell line. Overexpression of GAS8-AS1 inhibited proliferation, significantly increased the ratio of LC3-II/LC3-I, and reduced p62 expression, whereas GAS8-AS1 knockdown demonstrated opposite effects. In GAS8-AS1 overexpressing cell lines, LC3 immunofluorescence staining demonstrated increased punctate aggregates of LC3 staining, and transmission electron microscopy revealed increased numbers of autophagosomes. Autophagy-related gene 5 (ATG5) was markedly upregulated by GAS8-AS1 overexpression and downregulated by GAS8-AS1 knockdown. Finally, silencing of ATG5 attenuated autophagy activation and rescued the inhibition of cell proliferation caused by GAS8-AS1.
In PTC cell lines, GAS8-AS1 inhibited proliferation, activated autophagy, and increased ATG5 expression. Downregulation of ATG5 reversed GAS8-AS1-mediated activation of autophagy leading to cell death, revealing a novel mechanism of the GAS8-AS1-ATG5 axis in PTC cell lines. This provided a new experimental basis to explore the effects of lncRNA on autophagy in the treatment of thyroid cancer.
长链非编码 RNA GAS8 反义 RNA 1（lncRNA GAS8-AS1）是甲状腺乳头状癌（PTC）的肿瘤抑制因子，但 GAS8-AS1 调节 PTC 生物学的机制尚不清楚。在这里，我们评估了 GAS8-AS1 在调节 PTC 细胞系自噬中的分子功能。
通过转染表达质粒或小干扰 RNA（siRNA）在 PTC 细胞系中过表达和敲低 GAS8-AS1。使用细胞计数试剂盒-8（CCK-8）评估细胞增殖。qRT-PCR 和 Western blot 用于测定自噬相关基因表达的变化。通过免疫荧光和透射电子显微镜评估自噬。
与正常甲状腺细胞系相比，PTC 细胞系 TPC1 和 BCPAP 中的相对 GAS8-AS1 表达较低。GAS8-AS1 的过表达抑制增殖，显著增加 LC3-II/LC3-I 比值，并降低 p62 表达，而 GAS8-AS1 敲低则表现出相反的效果。在 GAS8-AS1 过表达细胞系中，LC3 免疫荧光染色显示 LC3 染色的点状聚集增加，透射电子显微镜显示自噬体数量增加。GAS8-AS1 过表达明显上调自噬相关基因 5（ATG5），GAS8-AS1 敲低下调 ATG5。最后，沉默 ATG5 减弱了自噬的激活并挽救了 GAS8-AS1 引起的细胞增殖抑制。
在 PTC 细胞系中，GAS8-AS1 抑制增殖，激活自噬，增加 ATG5 表达。下调 ATG5 逆转了 GAS8-AS1 介导的自噬激活导致的细胞死亡，揭示了 GAS8-AS1-ATG5 轴在 PTC 细胞系中的新机制。这为探索 lncRNA 对甲状腺癌自噬的影响提供了新的实验基础。
