Nucleostemin Knockdown Sensitizes Hepatocellular Carcinoma Cells to Ultraviolet and Serum Starvation-Induced Apoptosis

Nucleostemin (NS) is a GTP-binding protein that is predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS plays an essential role in maintaining the continuous proliferation of stem cells and some types of cancer cells. However, the role of NS in hepatocellular carcinoma (HCC) remains unclear. Therefore, this study aimed to clarify the role of NS in HCC. First, we demonstrated high expression of NS in most HCC cell lines and liver cancer tissues. NS knockdown induced a severe decline in cell viability of MHCC97H cells as detected by MTT and cell proliferation assays. Next, we used ultraviolet (UV) and serum starvation-induced apoptosis models to investigate whether NS suppression or up-regulation affects HCC cell apoptosis. After UV treatment or serum starvation, apoptosis was strongly enhanced in MHCC97H and Bel7402 cells transfected with small interfering RNA against NS, whereas NS overexpression inhibited UV- and serum-induced apoptosis of HCC cells. Furthermore, after UV irradiation, inhibition of NS increased the expression of pro-apoptosis protein caspase 3 and decreased the expression of anti-apoptosis protein Bcl-2. A caspase 3 inhibitor could obviously prevent NS knockdown-induced apoptosis. In conclusion, our study demonstrated overexpression of NS in most HCC tissues compared with their matched surrounding tissues, and silencing NS promoted UV- and serum starvation-induced apoptosis of MHCC97H and Bel7402 cells. Therefore, the NS gene might be a potential therapeutic target of HCC.

核仁素（Nucleostemin，NS）是一种GTP结合蛋白，主要在胚胎干细胞和成体干细胞中表达，而在终末分化细胞中无表达。NS在维持干细胞及部分癌细胞的持续增殖过程中发挥至关重要的作用，但目前其在肝细胞癌（hepatocellular carcinoma，HCC）中的具体功能仍不明确。为此，本研究旨在阐明NS在HCC中的作用机制。首先，我们证实多数HCC细胞系及肝癌组织中NS呈高表达水平；通过MTT法与细胞增殖实验检测发现，敲低NS可显著降低MHCC97H细胞的细胞活力。随后，我们构建了紫外线（ultraviolet，UV）照射及血清饥饿诱导的细胞凋亡模型，以探究NS敲低或过表达对HCC细胞凋亡的影响。结果显示，经UV照射或血清饥饿处理后，转染靶向NS的小干扰RNA（small interfering RNA）的MHCC97H与Bel7402细胞的凋亡水平显著增强；而NS过表达则可抑制UV照射及血清饥饿诱导的HCC细胞凋亡。此外，UV照射处理后，抑制NS的表达可上调促凋亡蛋白半胱天冬酶3（caspase 3）的表达，并下调抗凋亡蛋白B细胞淋巴瘤-2（Bcl-2）的表达；半胱天冬酶3抑制剂可显著阻断NS敲低诱导的细胞凋亡。综上，本研究证实，相较于配对的癌旁组织，多数HCC组织中NS呈现过表达状态；沉默NS可促进MHCC97H与Bel7402细胞在UV照射及血清饥饿诱导下的细胞凋亡，因此NS基因有望成为HCC潜在的治疗靶点。