A positive-feedback loop between HBx and ALKBH5 promotes hepatocellular carcinogenesis

Siming Qu,Zhong Zeng,Jie Lin,Hanfei Huang,Weiwu Gao,Li Jin

doi:10.1186/s12885-021-08449-5

Abstract

BackgroundHepatitis 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.MethodsThe 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 (m6A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.ResultALKBH5 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 m6A 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.ConclusionsHBx-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.

Highlights

Hepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms
Hepatitis B virus encode X protein (HBx)-AlkB Homolog 5 (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-Hepatocellular Carcinoma (HCC) treatment
Whether and how ALKBH5 is involved in HBV related HCC (HBVHCC) patients derived from Chinese people remains to be elucidated yet

Summary

Methods

Patients and HCC specimens This study was approved by the Ethical Committee of the First Affiliated Hospital of Kunming Medical University (Clinical Ethic Issue No: kmmu2019483) following guidelines of the Declaration of Helsinki. Twenty pairs of fresh HBV-HCC tissues with matched peri-tumor tissues from Chinese patients who were diagnosed as HBV-related primary HCC and normal liver tissues from patients with repair treatment of traumatic hepatic rupture were collected from the First Affiliated Hospital of Kunming Medical University to test the ALKBH5 mRNA and protein expression level. Final 1% concentration formaldehyde fixed chromatin from 10 million HepG2.2.15 cells with different treatment and were sonicated to about 500 bp fragments, antibody-bound protein A/G Dynabeads (Invitrogen) were incubated with sonicated chromatin at 4 °C overnight. Bioinformatics analysis RNA-seq and Mass spectrum (MS) data of 159 liver samples (paired tumor and peri-tumor) from Fudan Chinese HBV-HCC data was downloaded through the URL: https://www.biosino.org/node/project/detail/ OEP000321. Unpaired or paired two-tailed Student’s t-test were applied to assess comparison between two groups; Welch’s ANOVA test was used for comparison among groups; Kaplan-Meier survival curves and log-rank tests were used to estimate survival and differences between groups; The Pearson’s chi-square test was used for correlation analysis with calculation of correlation coefficients (r); The statistical significance was indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 and not significant (NS) when P > 0.05

Result

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