Elevated HMGB1 expression induced by hepatitis B virus X protein promotes epithelial-mesenchymal transition and angiogenesis through STAT3/miR-34a/NF-κB in primary liver cancer

Yuheng Zhang; Haozhen Ren; Jun Li; Ruifeng Xue; Hanyi Liu; Zhengyi Zhu; Chenyan Pan; Yunzhen Lin; Anyin Hu; Peng Gou; Jiahui Cai; Jingchao Zhou; Wei Zhu; Xiaolei Shi

Elevated HMGB1 expression induced by hepatitis B virus X protein promotes epithelial-mesenchymal transition and angiogenesis through STAT3/miR-34a/NF-κB in primary liver cancer

Am J Cancer Res. 2021 Feb 1;11(2):479-494. eCollection 2021.

Authors

Yuheng Zhang¹, Haozhen Ren¹, Jun Li², Ruifeng Xue¹, Hanyi Liu¹, Zhengyi Zhu¹, Chenyan Pan¹, Yunzhen Lin¹, Anyin Hu¹, Peng Gou¹, Jiahui Cai¹, Jingchao Zhou¹, Wei Zhu³, Xiaolei Shi¹

Affiliations

¹ Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School 321, Zhongshan Road, Nanjing 210008, Jiangsu, P. R. China.
² Department of General Surgery, The First Affiliated Hospital of Anhui Medical University Hefei 230022, Anhui, P. R. China.
³ Department of Anesthesiology, Affiliated Drum Tower Hospital of Nanjing University Medical School 321, Zhongshan Road, Nanjing 210008, Jiangsu, P. R. China.

PMID: 33575082
PMCID: PMC7868754

Abstract

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 in vivo metastatic experiments. We evaluated HMGB1 and STAT3/miR-34a/NF-κB pathway in vivo and in vitro 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.

Keywords: EMT; HBx; HMGB1; angiogenesis; liver cancer; miR-34a.