Zhou Yali, Zhao Yan, Gao Yaoying, Hu Wenjun, Qu Yan, Lou Ning, Zhu Ying, Zhang Xiaoping, Yang Hongmei
Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China.
Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei Province, China.
J Exp Clin Cancer Res. 2017 Mar 10;36(1):42. doi: 10.1186/s13046-017-0510-8.
Growing evidence suggests that hepatitis C virus (HCV) contributes to hepatocellular carcinoma (HCC) by directly modulating oncogenic signaling pathways. Protein phosphatase magnesium-dependent 1A (PPM1A) has recently emerged as an important tumor suppressor as it can block a range of tumor-centric signaling pathways through protein dephosphorylation. However, the role and regulatory mechanisms of PPM1A in HCV-infected cells have not been reported.
Total, cytoplasmic, and nuclear PPM1A protein after HCV infection or overexpression of HCV nonstructural protein 3 (NS3) were detected by western blotting. The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry. The effects of HCV infection and NS3 expression on the PPM1A protein level were systematically analyzed, and the ubiquitination level of PPM1A was determined by precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody. Finally, the roles of NS3 and PPM1A in hepatoma cell migration and invasion were assessed by wound healing and transwell assays, respectively.
HCV infection and replication decreased PPM1A abundance, mediated by NS3, in hepatoma cells. Compared to normal liver tissues, the expression of PPM1A was significantly decreased in the HCC tumor tissues and adjacent non-tumor tissues. NS3 directly interacted with PPM1A to promote PPM1A ubiquitination and degradation, which was dependent on its protease domain. Blockade of PPM1A through small interfering RNA significantly promoted HCC cell migration, invasion, and epithelial mesenchymal transition (EMT), which were further intensified by TGF-β1 stimulation, in vitro. Furthermore, restoration of PPM1A abrogated the NS3-mediated promotion of HCC migration and invasion to a great extent, which was dependent on its protein phosphatase function.
Our findings demonstrate that the HCV protein NS3 can downregulate PPM1A by promoting its ubiquitination and proteasomal degradation, which might contribute to the migration and invasion of hepatoma cells and may represent a new strategy of HCV in carcinogenesis.
越来越多的证据表明，丙型肝炎病毒（HCV）通过直接调节致癌信号通路促进肝细胞癌（HCC）的发生。蛋白磷酸酶镁依赖1A（PPM1A）最近已成为一种重要的肿瘤抑制因子，因为它可以通过蛋白质去磷酸化阻断一系列以肿瘤为中心的信号通路。然而，PPM1A在HCV感染细胞中的作用和调控机制尚未见报道。
通过蛋白质印迹法检测HCV感染或HCV非结构蛋白3（NS3）过表达后总的、细胞质和细胞核中的PPM1A蛋白。通过免疫组织化学法定量分析正常肝脏和HCV相关HCC组织中PPM1A的表达。系统分析HCV感染和NS3表达对PPM1A蛋白水平的影响，并通过抗PPM1A沉淀和抗泛素或抗PPM1A抗体免疫印迹法测定PPM1A的泛素化水平。最后，分别通过伤口愈合试验和Transwell试验评估NS3和PPM1A在肝癌细胞迁移和侵袭中的作用。
HCV感染和复制通过NS3介导降低了肝癌细胞中PPM1A的丰度。与正常肝脏组织相比，HCC肿瘤组织和相邻非肿瘤组织中PPM1A的表达明显降低。NS3直接与PPM1A相互作用，促进PPM1A的泛素化和降解，这依赖于其蛋白酶结构域。通过小干扰RNA阻断PPM1A在体外显著促进HCC细胞迁移、侵袭和上皮间质转化（EMT），TGF-β1刺激可进一步增强这些作用。此外，PPM1A的恢复在很大程度上消除了NS3介导的对HCC迁移和侵袭的促进作用，这依赖于其蛋白磷酸酶功能。
我们的研究结果表明，HCV蛋白NS3可通过促进PPM1A的泛素化和蛋白酶体降解而下调PPM1A，这可能有助于肝癌细胞的迁移和侵袭，并且可能代表了HCV在致癌过程中的一种新策略。
