Clinical Research Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, 100730, China.
Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, 100730, China.
Hepatol Int. 2019 Mar;13(2):180-189. doi: 10.1007/s12072-018-9920-8. Epub 2019 Jan 1.
Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues.
First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining.
The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial-mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson-Steiner grade, overall as well as tumor-free survival.
Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.
纤溶酶原激活物抑制剂 2（PAI2）已被证明与肝细胞癌（HCC）的侵袭表型和预后相关。然而，其在 HCC 侵袭中的生物学作用和潜在机制尚未得到探索。本研究旨在解决这些问题。
首先，基于 MHCC97H 和 BEL7402 细胞系，分别建立了 PAI2 稳定过表达和沉默的亚系。划痕愈合和 Transwell 分析用于评估细胞迁移和侵袭。酶联免疫吸附试验（ELISA）试剂盒用于测量尿激酶型纤溶酶原激活物（uPA）活性。实时 RT-PCR 和 Western blot 用于显示基因在 mRNA 和蛋白水平上的表达。通过组织微阵列免疫组织化学染色确定人标本中的 E2F1 表达。
成功建立了 MHCC97H-PAI2 和 BEL7402-siPAI2 亚系。与对照相比，这两个亚系的迁移和侵袭能力分别明显降低和增强。在 MHCC97H-PAI2 亚系中，与对照相比，细胞培养基中的 uPA 活性显著降低，而 RB 表达明显升高。BEL7402-siPAI2 亚系则呈现相反的趋势。为了确定 RB/E2F1 通路的作用，我们在 MHCC97H-PAI2 亚系中转染瞬时过表达 E2F1，通过调节多种基质金属蛋白酶和上皮-间充质转化，在很大程度上逆转了 PAI2 对细胞迁移和侵袭的抑制作用。在 HCC 标本中，E2F1 表达在肿瘤组织中明显高于非肿瘤组织，且与 Edmondson-Steiner 分级、总生存率和无瘤生存率显著相关。
我们的数据表明，PAI2 通过 uPA 和 RB/E2F1 相关机制抑制 HCC 细胞的侵袭潜力。
