HMGB1-mediated autophagy regulates sodium/iodide symporter protein degradation in thyroid cancer cells
HMGB1-mediated autophagy regulates sodium/iodide symporter protein degradation in thyroid cancer cells
复制标题
HMGB1介导的自噬调节甲状腺癌细胞中钠/碘同向转运蛋白的降解
DOI：
10.1186/s13046-019-1328-3
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发表时间：
2019-07-22
影响因子：
11.3
通讯作者：
Yang, Liangchun
中科院分区：
文献类型：
--
作者：
Chai, Wenwen;Ye, Fanghua;Yang, Liangchun
研究背景钠/碘转运体（NIS）介导的碘摄取在甲状腺功能调节、Graves病和甲状腺癌的诊断和治疗中起着重要的生理作用。高迁移率族蛋白1（HMGB 1）是一种高度保守的核蛋白，是自噬的正调控因子，可使癌细胞对化疗、放疗和免疫治疗产生抗性。本研究旨在探讨HMGB 1在Hank平衡盐溶液（HBSS）诱导的甲状腺癌细胞自噬中的作用，探讨NIS蛋白通过自噬-溶酶体途径降解的可能机制。通过慢病毒转染FTC-133/TPC-1细胞，HMGB 1被敲低。自噬标志物LC 3-II、p62、Beclin 1和自噬体形成通过Western blot、免疫荧光和电子显微镜来评价HMGB 1介导的HBSS处理的细胞中的自噬。采用Western blot、定量RT-PCR和γ计数法检测HMGB 1基因敲除细胞经不同处理后NIS表达和碘摄取的变化。采用荧光分光光度计、流式细胞仪、Western blot和免疫荧光法检测细胞内活性氧（ROS）水平、ROS介导的LC 3-II表达和HMGB 1胞浆转位。Western blot检测HMGB 1介导的AMPK、mTOR和p70 S6 K磷酸化（p-AMPK、p-mTOR和p-p70 S6 K）。此外，采用移植肿瘤的裸小鼠模型来检查HMGB 1介导的自噬对（99 m）TcO 4（-）成像和生物分布的影响。结果HMGB 1是HBSS诱导FTC-133/TPC-1细胞自噬过程中NIS降解的重要调控因子。HMGB 1的上调在甲状腺癌组织中相当普遍，并且与整体淋巴结转移和临床分期密切相关。HMGB 1敲低显著抑制了HBSS处理的细胞中的自噬、NIS降解并促进了碘化物摄取。此外，HBSS增强ROS持续的自噬，并促进HMGB 1的胞质转位。HMGB 1的敲低通过AMPK/mTOR依赖性信号通路抑制LC 3-II转化和NIS降解，通过调节ROS产生，而不是ATP。结论HMGB 1通过ROS/AMPK/mTOR途径调控自噬介导的NIS降解，是甲状腺癌放射性碘治疗的潜在干预靶点。
BackgroundSodium/iodide symporter (NIS)-mediated iodide uptake plays an important physiological role in regulating thyroid gland function, as well as in diagnosing and treating Graves' disease and thyroid cancer. High-mobility group box1 (HMGB1), a highly conserved nuclear protein, is a positive regulator of autophagy conferring resistance to chemotherapy, radiotherapy and immunotherapy in cancer cells. Here the authors intended to identify the role of HMGB1 in Hank's balanced salt solution (HBSS)-induced autophagy, explore NIS protein degradation through a autophagy-lysosome pathway in thyroid cancer cells and elucidate the possible molecular mechanisms.MethodsImmunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) were performed for detecting the expression of HMGB1 in different tissues. HMGB1 was knocked down by lentiviral transfection in FTC-133/TPC-1 cells. Autophagic markers LC3-II, p62, Beclin1 and autophagosomal formation were employed for evaluating HMGB1-mediated autophagy in HBSS-treated cells by Western blot, immunofluorescence and electron microscopy. Western blot, quantitative RT-PCR and gamma counter analysis were performed for detecting NIS expression and iodide uptake in HMGB1-knockdown cells after different treatments. The reactive oxygen species (ROS) level, ROS-mediated LC3-II expression and HMGB1 cytosolic translocation were detected by fluorospectrophotometer, flow cytometry, Western blot and immunofluorescence. HMGB1-mediated AMPK, mTOR and p70S6K phosphorylation (p-AMPK, p-mTOR & p-p70S6K) were detected by Western blot. Furthermore, a nude murine model with transplanted tumor was employed for examining the effect of HMGB1-mediated autophagy on imaging and biodistribution of (99m)TcO4(-). NIS, Beclin1, p-AMPK and p-mTOR were detected by immunohistochemical staining and Western blot in transplanted tumor samples.ResultsHMGB1 was a critical regulator of autophagy-mediated NIS degradation in HBSS-treated FTC-133/TPC-1 cells. And HMGB1 up-regulation was rather prevalent in thyroid cancer tissues and closely correlated with worse overall lymph node metastasis and clinical stage. HMGB1-knockdown dramatically suppressed autophagy, NIS degradation and boosted iodide uptake in HBSS-treated cells. Moreover, HBSS enhanced ROS-sustained autophagy and promoted the cytosolic translocation of HMGB1. A knockdown of HMGB1 suppressed LC3-II conversion and NIS degradation via an AMPK/mTOR-dependent signal pathway through a regulation of ROS generation, rather than ATP. Furthermore, these data were further supported by our in vivo experiment of xenografts formed by HMGB1 knockdown cells reverting the uptake of (99m)TcO4(-) as compared with control shRNA-transfected cells in hunger group.ConclusionsActing as a critical regulator of autophagy-mediated NIS degradation via ROS/AMPK/mTOR pathway, HMGB1is a potential intervention target of radioiodine therapy in thyroid cancer.