Organ Transplantation Center, the First Affiliated Hospital of Kunming Medical University, 295 Xichang Road, Kunming, 650032, Yunnan Province, China.
Institute of Immunology, PLA, Third Military Medical University, 30 Gaotanyan St., District Shapingba, Chongqing, 400038, China.
BMC Cancer. 2021 Jun 10;21(1):686. doi: 10.1186/s12885-021-08449-5.
Hepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms. The newly defined epigenetics, epitranscriptomics regulation, has been reported to involve in multiple cancers including Hepatocellular Carcinoma (HCC). Our previous study found that HBx, HBV encodes X protein, mediated H3K4me3 modification in WDR5-dependent manner to involve in HBV infection and contribute to oncogene expression. AlkB Homolog 5 (ALKBH5), one of epitranscriptomics enzymes, has been identified to be associated with various cancers. However, whether and how ALKBH5 is dysregulated in HBV-related HCC remains unclear yet. This study aims to investigate ALKBH5 function, clinical significance and mechanism in HBV related HCC (HBV-HCC) patients derived from Chinese people.
The expression pattern of ALKBH5 was evaluated by RT-qPCR, Western blot, data mining and immunohistochemistry in total of 373 HBV-HCC tissues and four HCC cell lines. Cell Counting Kit 8 (CCK8) assay, Transwell and nude mouse model were performed to assess ALKBH5 function by both small interference RNAs and lentiviral particles. The regulation mechanism of ALKBH5 was determined in HBx and WDR5 knockdown cells by CHIP-qPCR. The role of ALKBH5 in HBx mRNA N6-methyladenosine (mA) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.
ALKBH5 increased in tumor tissues and predicts a poor prognosis of HBV-HCC. Mechanically, the highly expressed ALKBH5 is induced by HBx-mediated H3K4me3 modification of ALKBH5 gene promoter in a WDR5-dependent manner after HBV infection. The increased ALKBH5 protein catalyzes the mA demethylation of HBx mRNA, thus stabilizing and favoring a higher HBx expression level. Furthermore, there are positive correlations between HBx and ALKBH5 in HBV-HCC tissues, and depletion of ALKBH5 significantly inhibits HBV-driven tumor cells' growth and migration in vitro and in vivo.
HBx-ALKBH5 may form a positive-feedback loop to involve in the HBV-induced liver carcinogenesis, and targeting the loop at ALKBH5 may provide a potential way for HBV-HCC treatment.
乙型肝炎病毒 (HBV) 通过多种表观遗传机制促进肝癌的发生。新定义的表观遗传学，即转录后修饰调控，已被报道参与多种癌症，包括肝细胞癌 (HCC)。我们之前的研究发现，HBx，HBV 编码的 X 蛋白，通过 WDR5 依赖性方式介导 H3K4me3 修饰，参与 HBV 感染，并有助于癌基因表达。ALKBH5，一种转录后修饰酶，已被确定与多种癌症相关。然而，ALKBH5 在 HBV 相关 HCC 中的表达是否失调以及如何失调尚不清楚。本研究旨在探讨 ALKBH5 在中国人来源的 HBV 相关 HCC (HBV-HCC) 患者中的功能、临床意义和机制。
通过 RT-qPCR、Western blot、数据挖掘和免疫组织化学方法，共检测了 373 例 HBV-HCC 组织和 4 种 HCC 细胞系中 ALKBH5 的表达模式。通过小干扰 RNA 和慢病毒颗粒进行细胞计数试剂盒 8 (CCK8) 检测、Transwell 和裸鼠模型，以评估 ALKBH5 的功能。通过 CHIP-qPCR 在 HBx 和 WDR5 敲低细胞中确定 ALKBH5 的调节机制。通过 MeRIP-qPCR 和 Actinomycin D 抑制剂实验，进一步评估 ALKBH5 在 HBV 驱动的细胞和 HBx 过表达细胞中对 HBx mRNA N6-甲基腺苷 (mA) 修饰的作用。
ALKBH5 在肿瘤组织中增加，并预测 HBV-HCC 的预后不良。从机制上讲，HBx 感染后，HBx 介导的 ALKBH5 基因启动子 H3K4me3 修饰以 WDR5 依赖的方式诱导高表达的 ALKBH5。增加的 ALKBH5 蛋白催化 HBx mRNA 的 mA 去甲基化，从而稳定并有利于更高的 HBx 表达水平。此外，HBx 和 ALKBH5 在 HBV-HCC 组织中存在正相关，ALKBH5 的耗竭可显著抑制 HBV 驱动的肿瘤细胞在体外和体内的生长和迁移。
HBx-ALKBH5 可能形成正反馈环，参与 HBV 诱导的肝癌发生，针对 ALKBH5 的靶向治疗可能为 HBV-HCC 的治疗提供一种潜在的方法。
