Yoo Jeong-Ju, Lee Dong Hyeon, Cho Yuri, Cho Eun Ju, Lee Jeong-Hoon, Yu Su Jong, Kim Yoon Jun, Kim Chung Yong, Yoon Jung-Hwan
Department of Gastroenterology and Hepatology, Soonchunhyang University Bucheon Hospital, Gyeonggi do, Republic of Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
J Bioenerg Biomembr. 2016 Dec;48(6):581-590. doi: 10.1007/s10863-016-9677-5. Epub 2016 Sep 17.
Mutations in the gene encoding hepatocystin/80 K-H (PRKCSH) cause autosomal dominant polycystic liver disease. Hepatocystin deficiency impairs glucosidase II activity, which is critical for processing and folding glycoproteins in the endoplasmic reticulum (ER). Hypoxia is known as a strong stimulus for generating survival signals in hepatocellular carcinoma (HCC) cells. However, hypoxia may induce cell apoptosis under conditions of severe ER stress. Thus, we hypothesized that suppression of hepatocystin transcription induces HCC cell death under hypoxic conditions due to excessive ER stress. A new human HCC cell line, SNU-3058, was established following primary culture of tumor cells harvested from a Korean patient with rapidly growing hypovascular HCC. In cell culture, human HCC cells (Huh-7, SNU-761, and SNU-3058) were treated with control siRNA or hepatocystin siRNA with or without doxorubicin under hypoxic conditions. Cell viability, ER stress, unfolded protein response (UPR), and apoptosis were assessed using the MTS assay, immunoblot assay, and RT-PCR. Suppression of hepatocystin transcription attenuated proliferation in Huh-7 and SNU-761 cells, while proliferation was amplified in SNU-3058 cells. Similar results were observed following treatment with doxorubicin. Hepatocystin siRNA transfection increased cell death in Huh-7 and decreased cell death in SNU-3058. In SNU-3058, hepatocystin siRNA amplified GRP78, known as a pro-survival and cyto-protective signal, and attenuated the pro-apoptotic signal CHOP. These findings suggest that suppression of hepatocystin transcription induce the UPR, which alleviates damage associated with ER stress in SNU-3058. UPR had a limited role in protecting SNU-761 cells, resulting in cell death through apoptosis. In addition, blocking of pro-survival UPR signal by bacitracin or GRP78 knockdown, attenuated hepatocystin siRNA-induced proliferation in SNU-3058 cells under hypoxia. In this study, we demonstrated that different sensitivities to hepatocystin siRNA among human HCC cell lines are dependent on appropriate UPRs to hypoxia-induced ER stress following hepatocystin siRNA transfection. Because UPR is the main evasive mechanism for apoptosis induced by suppression of hepatocystin, targeting hepatocystin via UPR suppression could be a strategy for treating HCC.
编码肝囊肿素/80K-H（PRKCSH）的基因突变会导致常染色体显性多囊肝病。肝囊肿素缺乏会损害葡萄糖苷酶II的活性，而该酶对于内质网（ER）中糖蛋白的加工和折叠至关重要。缺氧是肝细胞癌（HCC）细胞中产生生存信号的强烈刺激因素。然而，在严重内质网应激条件下，缺氧可能诱导细胞凋亡。因此，我们推测在缺氧条件下，由于内质网应激过度，肝囊肿素转录的抑制会诱导肝癌细胞死亡。从一名患有快速生长的乏血供肝癌的韩国患者身上采集肿瘤细胞进行原代培养后，建立了一种新的人肝癌细胞系SNU-3058。在细胞培养中，人肝癌细胞（Huh-7、SNU-761和SNU-3058）在缺氧条件下用对照小干扰RNA（siRNA）或肝囊肿素siRNA处理，同时添加或不添加阿霉素。使用MTS检测法、免疫印迹检测法和逆转录聚合酶链反应（RT-PCR）评估细胞活力、内质网应激、未折叠蛋白反应（UPR）和细胞凋亡。肝囊肿素转录的抑制减弱了Huh-7和SNU-761细胞的增殖，而SNU-3058细胞的增殖则增强。用阿霉素处理后也观察到了类似结果。肝囊肿素siRNA转染增加了Huh-7细胞的死亡，减少了SNU-3058细胞的死亡。在SNU-3058细胞中，肝囊肿素siRNA增强了GRP78（一种促生存和细胞保护信号）的表达，并减弱了促凋亡信号CHOP的表达。这些发现表明，肝囊肿素转录的抑制会诱导未折叠蛋白反应，从而减轻SNU-3058细胞中与内质网应激相关的损伤。未折叠蛋白反应在保护SNU-761细胞方面作用有限，导致细胞通过凋亡死亡。此外，杆菌肽或GRP78基因敲低对促生存未折叠蛋白反应信号的阻断，减弱了缺氧条件下肝囊肿素siRNA诱导的SNU-3058细胞增殖。在本研究中，我们证明了人肝癌细胞系对肝囊肿素siRNA的不同敏感性取决于肝囊肿素siRNA转染后对缺氧诱导的内质网应激的适当未折叠蛋白反应。由于未折叠蛋白反应是肝囊肿素抑制诱导凋亡的主要逃避机制，通过抑制未折叠蛋白反应靶向肝囊肿素可能是一种治疗肝癌的策略。
