Zhang Yuheng, Ren Haozhen, Li Jun, Xue Ruifeng, Liu Hanyi, Zhu Zhengyi, Pan Chenyan, Lin Yunzhen, Hu Anyin, Gou Peng, Cai Jiahui, Zhou Jingchao, Zhu Wei, Shi Xiaolei
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School 321, Zhongshan Road, Nanjing 210008, Jiangsu, P. R. China.
Department of General Surgery, The First Affiliated Hospital of Anhui Medical University Hefei 230022, Anhui, P. R. China.
Am J Cancer Res. 2021 Feb 1;11(2):479-494. eCollection 2021.
HBV infection plays a crucial role in primary liver cancer development. Also, HBV related liver cancer has higher invasiveness and earlier discovered distant metastasis. HBV-encoded X protein (HBx) exerts various biological functions on liver cancer progression, including proliferation, invasion, and venous metastasis. There is evidence that High-mobility group box 1 (HMGB1) promotes epithelial-mesenchymal transition (EMT) and angiogenesis of tumors, including liver cancer. Therefore, this study investigates whether HMGB1 mediates HBx-induced EMT and angiogenesis in HBV related liver cancer. We collected 76 tumor samples of primary liver cancer patients to analyze the relationship between HMGB1 and portal vein tumor thrombus (PVTT) in HBV related liver cancer. To test the influence of HMGB1 on EMT and angiogenesis, we constructed HBx lentivirus transfected HepG2/Huh7 cell lines and performed invasion assays, tube formation and metastatic experiments. We evaluated HMGB1 and STAT3/miR-34a/NF-κB pathway and by immunoblot, quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence and immunohistochemistry analysis. Subsequent RNA interference (RNAi) and luciferase reporter assay were conducted to detect the functional correlation between HMGB1 and STAT3/miR-34a/NF-κB pathway. Our results showed enhanced expression of HMGB1 in HBV related liver cancer, especially with PVTT, while HMGB1 expression was associated with tumor invasion and metastasis. Further experiments indicated that the activation of STAT3 mediated HBx-induced HMGB1, which is involved in EMT and tumor angiogenesis. Besides, HMGB1 expression stimulated by HBx was dependent on the activation of the NF-κB signaling pathway, which was inhibited by miR-34a, while STAT3 suppressed the expression of miR-34a. Moreover, extracellular HMGB1 induced the IL-6/STAT3/miR-34a axis activation, which indicated a reciprocal relationship between HMGB1 and miR-34a. Collectively, our study provided evidence to reveal that HBx-mediated high expression of HMGB1 accounted for EMT and tumor angiogenesis in HBV related liver cancer, and HMGB1 may be a potential target for predicting venous metastasis.
乙肝病毒（HBV）感染在原发性肝癌的发生发展中起着关键作用。此外，HBV相关肝癌具有更高的侵袭性，且远处转移发现得更早。HBV编码的X蛋白（HBx）对肝癌进展发挥多种生物学功能，包括增殖、侵袭和静脉转移。有证据表明，高迁移率族蛋白B1（HMGB1）促进包括肝癌在内的肿瘤上皮-间质转化（EMT）和血管生成。因此，本研究探讨HMGB1是否介导HBx诱导的HBV相关肝癌中的EMT和血管生成。我们收集了76例原发性肝癌患者的肿瘤样本，以分析HMGB1与HBV相关肝癌中门静脉癌栓（PVTT）之间的关系。为了测试HMGB1对EMT和血管生成的影响，我们构建了HBx慢病毒转染的HepG2/Huh7细胞系，并进行侵袭实验、管腔形成实验和转移实验。我们通过免疫印迹、定量实时聚合酶链反应（qRT-PCR）、免疫荧光和免疫组织化学分析评估了HMGB1和STAT3/miR-34a/NF-κB通路。随后进行RNA干扰（RNAi）和荧光素酶报告基因检测，以检测HMGB1与STAT3/miR-34a/NF-κB通路之间的功能相关性。我们的结果显示，HMGB1在HBV相关肝癌中表达增强，尤其是在伴有PVTT的情况下，而HMGB1的表达与肿瘤侵袭和转移相关。进一步的实验表明，STAT3的激活介导了HBx诱导的HMGB1，其参与了EMT和肿瘤血管生成。此外，HBx刺激的HMGB1表达依赖于NF-κB信号通路的激活，而该通路被miR-34a抑制，同时STAT3抑制miR-34a的表达。此外，细胞外HMGB1诱导IL-6/STAT3/miR-34a轴激活，这表明HMGB1与miR-34a之间存在相互关系。总的来说，我们的研究提供了证据，揭示HBx介导的HMGB1高表达导致HBV相关肝癌中的EMT和肿瘤血管生成，并且HMGB1可能是预测静脉转移的潜在靶点。
