Data Sheet 1_Distinct roles of Constitutive Photomorphogenesis Protein 1 homolog (COP1) in human hepatocyte models.pdf

IntroductionConstitutive Photomorphogenesis Protein 1 homolog (COP1) is a conserved E3 ligase with key roles in several biological systems. Prior work in hepatocyte-derived tumors categorized COP1 as an oncogene, but its role in untransformed hepatocytes remains largely unexplored. Here, we have investigated the role of COP1 in primary human hepatocytes and two transformed hepatocyte models, HepG2 and HuH-7 cells.
MethodsThe role of COP1 was tested by silencing and transduction experiments in HepG2, HuH-7, and primary human hepatocytes. Transcription array data of COP1-suppressed cells were generated and analyzed using clustering analyses. Cellular impacts were examined by proliferation assays, qRT-PCR, western blotting, reporter assays, and APOB enzyme-linked immunosorbent assays.
Results and DiscussionCOP1 suppression had no noticeable impact on HepG2 and HuH-7 proliferation and was associated with contrasting rather than congruent transcriptome changes. Transcriptomic changes were consistent with perturbed metabolism in primary hepatocytes and HepG2 cells and impaired cell cycle regulation in HuH-7 cells. In HepG2 and primary hepatocytes but not in HuH-7 cells, COP1 suppression reduced the expression of important hepatic regulators and markers. COP1 downregulation reduced hepatic nuclear factor-4 alpha (HNF4A) abundance and function, as assessed by a lower abundance of key HNF4A targets, reduced APOB secretion, and reporter assays. HNF4A function could be restored by introducing a siRNA-resistant COP1 transgene, whereas HNF4A restoration partially rescued COP1 silencing in HepG2 cells. Our results identify and detail a pivotal regulatory role of COP1 in hepatocytes, in part through HNF4A.

引言
组成型光形态发生蛋白1同源物（Constitutive Photomorphogenesis Protein 1 homolog，COP1）是一种保守的E3泛素连接酶（E3 ligase），在多种生物系统中发挥关键作用。既往针对肝细胞源性肿瘤的研究将COP1归类为癌基因（oncogene），但其在未转化肝细胞中的功能仍未得到充分探索。本研究针对原代人肝细胞以及两种转化肝细胞模型（HepG2与HuH-7细胞）中COP1的功能展开了探究。

方法
本研究通过基因沉默与转导实验，在HepG2、HuH-7细胞及原代人肝细胞中验证了COP1的功能。本研究获取了COP1沉默细胞的转录组芯片数据，并通过聚类分析开展相关分析。通过增殖实验、实时定量反转录聚合酶链式反应（qRT-PCR）、蛋白质免疫印迹（western blotting）、报告基因实验以及载脂蛋白B（APOB）酶联免疫吸附实验（ELISA），对细胞层面的生物学效应进行了检测。

结果与讨论
沉默COP1对HepG2与HuH-7细胞的增殖无显著影响，且其诱导的转录组变化呈现出分化而非趋同的特征。转录组学分析结果显示，原代肝细胞与HepG2细胞的代谢通路受到扰动，而HuH-7细胞则存在细胞周期调控缺陷。在HepG2细胞与原代肝细胞中，沉默COP1会降低重要肝脏调控因子及标志物的表达水平，但该效应并未出现在HuH-7细胞中。通过检测关键肝细胞核因子4α（hepatic nuclear factor-4 alpha, HNF4A）靶标的丰度下降、APOB分泌减少以及报告基因实验结果，证实COP1下调会降低HNF4A的表达水平与功能活性。通过转入对小干扰RNA（small interfering RNA, siRNA）耐受的COP1转基因，可恢复HNF4A的功能；而在HepG2细胞中，恢复HNF4A的表达可部分逆转COP1沉默带来的效应。本研究明确并详细阐释了COP1在肝细胞中发挥的关键调控功能，且该功能部分依赖于HNF4A通路。