Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin Geriatrics Institute, Tianjin 300052, China
Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin Geriatrics Institute, Tianjin 300052, China.
Biosci Rep. 2017 Oct 31;37(5). doi: 10.1042/BSR20170114.
Thyroid cancer patients with high miR-490-3p inhibit translation of mRNA, whereas in patients with low miR-490-3p mRNA expression is high; however, the resultant protein is targeted for degradation through the proteasome. The objective of the present study was to evaluate the molecular mechanism that regulates post-translation degradation of poly r(C) binding protein (PCBP) 1 expression in thyroid cancer cells. Mass spectrometric analysis of PCBP1 immunoprecipitates from MG-132 treated TPC1 cells revealed a list of ubiquitin ligases associated with PCBP1. RNAi-mediated silencing of the candidate ubiquitin ligases revealed that knockdown of the ubiquitin ligase stabilized PCBP1 in TPC1 cells. Concurrent overexpression of the candidate ubiquitin ligases in the normal thyroid epithelial cell line Nthy-ori 3-1 confirmed that ubiquitin conjugation factor E4 A (UBE4A) is the ubiquitin ligase that is degrading PCBP1. Coimmunoprecipitation of HA-tagged PCBP1 in TPC1 cells cotransfected with FLAG- revealed robust polyubiquitinated smear of PCBP1, thus confirming UBE4A as the ubiquitin ligase of PCBP1. expression mimicked mRNA expression in thyroid cancer patients and was inversely correlated to PCBP1 protein expression. Low expression level was associated with a better prognosis in thyroid cancer patients. Our data reveal a post-translational regulatory mechanism of regulating PCBP1 expression in thyroid cancer cells.
甲状腺癌患者中高表达 miR-490-3p 会抑制 mRNAs 的翻译，而低表达 miR-490-3p 的患者中 mRNAs 的表达水平较高；然而，翻译后的蛋白质会通过蛋白酶体靶向降解。本研究旨在评估调节甲状腺癌细胞中多聚 r(C)结合蛋白 1 (PCBP1)表达的翻译后降解的分子机制。用 MG-132 处理 TPC1 细胞后，用 PCBP1 免疫沉淀进行质谱分析，揭示了与 PCBP1 相关的一系列泛素连接酶。候选泛素连接酶的 RNAi 介导沉默显示，泛素连接酶的敲低可稳定 TPC1 细胞中的 PCBP1。候选泛素连接酶在正常甲状腺上皮细胞系 Nthy-ori 3-1 中的过表达同时证实，泛素连接酶 E4 A (UBE4A)是降解 PCBP1 的泛素连接酶。在共转染 FLAG-的 TPC1 细胞中，HA 标记的 PCBP1 的共免疫沉淀显示 PCBP1 有强烈的多聚泛素化斑点，从而证实 UBE4A 是 PCBP1 的泛素连接酶。在甲状腺癌患者中， 表达模拟了 mRNAs 的表达，与 PCBP1 蛋白表达呈负相关。低 表达水平与甲状腺癌患者的较好预后相关。我们的数据揭示了调节甲状腺癌细胞中 PCBP1 表达的一种翻译后调控机制。
