The Second Affiliated Hospital and the Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China.
Int J Cancer. 2014 Aug 1;135(3):635-46. doi: 10.1002/ijc.28697. Epub 2014 Jan 13.
Secreted frizzled-related proteins (SFRPs) are antagonists of the Wnt signaling pathway whose epigenetic downregulation have been shown to be involved in hepatocarcinogenesis. However, dysregulation of SFRPs induced by hepatitis B virus (HBV) X protein (HBx) has never been studied in HBV-related hepatocellular carcinoma (HBV-HCC). In this study, we sought to determine the clinical significance and underlying mechanism of HBx-induced SFRPs dysregulation in hepatoma cells and HBV-HCC patients. Our results showed that SFRP1 and SFRP5 expression were dramatically decreased by HBx in hepatoma cells. The repressed expression in hepatoma cells was partially rescued by a DNA methylation inhibitor and synergistically increased by a combination treatment with a histone deacetyltransferases inhibitor. In addition, we identified that SFRP1 and SFRP5 promoters were hypermethylated in both HBx-expressing hepatoma cells and HBV-HCC tissues. Downregulation of SFRP1 and SFRP5 in HBV-HCC tissues was significantly correlated with overexpression of DNA methyltransferase 1 (DNMT1) and poor tumor differentiation. HBx facilitated the binding of DNMT1 and DNMT3A to SFRP1 and SFRP5 promoters, and resulted in epigenetic silencing of SFRP1 and SFRP5. Moreover, overexpression of SFRP1, SFRP5 or RNA interference mediated silencing of DNMT1 inactivated the Wnt signaling pathway and decreased the expression levels of Wnt target genes c-Myc and CyclinD1, thus impeding HCC growth in vitro and in vivo, and regressing HBx-induced epithelial-mesenchymal transition (EMT). Our findings strongly suggest that epigenetic silencing of SFRP1 and SFRP5 by HBx allows constitutive activation of Wnt signaling pathway and hence contributes to hepatocarcinogenesis.
分泌型卷曲相关蛋白（SFRPs）是 Wnt 信号通路的拮抗剂，其表观遗传下调已被证明与肝癌发生有关。然而，乙型肝炎病毒（HBV）X 蛋白（HBx）诱导的 SFRPs 失调在 HBV 相关肝细胞癌（HBV-HCC）中从未被研究过。在这项研究中，我们试图确定 HBx 在肝癌细胞和 HBV-HCC 患者中诱导 SFRPs 失调的临床意义和潜在机制。我们的结果表明，HBx 可显著降低肝癌细胞中 SFRP1 和 SFRP5 的表达。在肝癌细胞中，DNA 甲基化抑制剂部分挽救了这种抑制作用，与组蛋白去乙酰化酶抑制剂联合治疗协同增加。此外，我们还发现，HBx 表达的肝癌细胞和 HBV-HCC 组织中 SFRP1 和 SFRP5 启动子均发生超甲基化。HBV-HCC 组织中 SFRP1 和 SFRP5 的下调与 DNA 甲基转移酶 1（DNMT1）的过度表达和肿瘤分化不良显著相关。HBx 促进了 DNMT1 和 DNMT3A 与 SFRP1 和 SFRP5 启动子的结合，导致 SFRP1 和 SFRP5 的表观遗传沉默。此外，SFRP1、SFRP5 的过表达或 RNA 干扰介导的 DNMT1 沉默使 Wnt 信号通路失活，降低了 Wnt 靶基因 c-Myc 和 CyclinD1 的表达水平，从而抑制 HCC 在体外和体内的生长，并使 HBx 诱导的上皮-间充质转化（EMT）逆转。我们的研究结果强烈表明，HBx 对 SFRP1 和 SFRP5 的表观遗传沉默允许 Wnt 信号通路的持续激活，从而促进肝癌发生。
