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

Abstract

Background: 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.

Methods: 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 (m⁶A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.

Result: 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 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.

Conclusions: 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.

Keywords: ALKBH5; Epitranscriptomics; HBx; HCC; m6A modification.

Fig. 1

The mRNA and proterin level of ALKBH5 are highly expressed in HBV-HCC tissue. AThe mRNA and B protein levels of ALKBH5 in the indicated HBV-HCC or matched peri-tumor tissues were determined by RT-qPCR (n = 20) and Western blot assays (n = 20), respectively. GAPDH was used as the internal control in RT-qPCR assay and β-actin was used as the normalized control in Western blotting assay. C Representative images of ALKBH5 IHC staining in normal liver, peri-tumor and tumor tissue of HBV-HCC patients (magnification 200×). D ALKBH5 IHC score in tumor and paired peri-tumor from 79 HBV-HCC tissue microarray. E Kaplan-Meier analysis of ALKBH5 IHC score and overall survival from 79 HBV-HCC tissue microarray. T, tumor; P, peri-tumor

Fig. 2

ALKBH5 is predominately increased in 274 HBV-HCC patients and predicts poor prognosis. A, B ALKBH5 mRNA TPM score and protein abundance in tumor and paired peri-tumor from 159 Fudan HBV-HCC patients’ transcriptome and proteome dataset. C ALKBH5 mRNA TPM score in tumors of Asian, Black and White ethnicity from 115 TCGA HBV-HCC patients’ transcriptome. D ALKBH5 mRNA TPM score in tumors of HBV C, B and other genotypes from 115 TCGA HBV-HCC patients’ transcriptome. E Kaplan-Meier analysis of ALKBH5 mRNA TPM, protein abundance and overall survival of 159 Fudan HBV-HCC patients. F Kaplan-Meier analysis of ALKBH5 mRNA TPM and overall survival of 115 TCGA HBV-HCC patients. T, tumor; P, peri-tumor

Fig. 3

HBx up-regulates ALKBH5. A The protein level of ALKBH5 in the indicated cell lines. B ALKBH5 protein level in PHH cells with or without HBV WT or HBV MT virion infection. C ALKBH5 protein level in HepG2-NTCP cells with or without HBV WT or HBV MT virion infection. D Correlation analysis between HBx and ALKBH5 mRNA expression determined by RT-qPCR in tumors from 24 HBV-HCC patients. E, F ALKBH5 mRNA and protein levels in HepG2 cells with or without HBx transduction. WT, widetype; MT, mutant

Fig. 4

HBV up-regulates ALKBH5 via the HBx-WDR5-H3K4me3 axis. A HBx, WDR5 and H3K4me3 distribution at the ALKBH5 gene locus. Snapshot generated by the UCSC Genome Browser. B The H3K4me3 level of ALKBH5 promoter was measured by ChIP-qPCR after either HBx or WDR5 knockdown compared to the control. C WDR5 mRNA TPM score and protein abundance in tumor and paired peri-tumor from 159 Fudan HBV-HCC patients’ transcriptome and proteome dataset. D Left panel: Kaplan-Meier analysis of WDR5 mRNA TPM, protein abundance and overall survival of 159 Fudan HBV-HCC patients. Right panel: Correlation analysis between WDR5 and ALKBH5 mRNA TPM, protein abundance from 159 Fudan HBV-HCC patients’ transcriptome and proteome dataset. E WDR5 protein level in HepG2.2.15 cells with or without ALKBH5 knockdown. T, tumor; P, peri-tumor

Fig. 5

ALKBH5 promotes the growth and migration of hepatoma cells. A Efficiency of ALKBH5 knockdown (KD) in HepG2 and HepG2.2.15 cells were evaluated by Western blot assay. B Proliferation rates of HepG2 and HepG2.2.15 with or without silencing of ALKBH5 were detected by CCK8 assay. C Effect of knockdown of ALKBH5 on migration rates of HepG2 and HepG2.2.15 were detected by transwell assay. D, E Tumor size and weight in each group. 1 × 10⁷ of HepG2.2.15 cells (control) or HepG2.2.15 cells with ALKBH5-knockdown (KD) were subcutaneously injected into nude mice, and the tumor size (D) and weight (E) were measured 3 weeks after tumor cells inoculation

Fig. 6

ALKBH5 favors HBx mRNA stability by regulating its m⁶A modification. A Map of m⁶A modification sites in the HBV ayw genome by MeRIP-seq of polyA-RNA isolated from HepG2.2.15 cells. Read coverage, normalized to the total number of reads mapping to the viral genome, is in green for MeRIP-seq and in yellow for input RNA-seq. The major m⁶A peak on HBV genome in the red box. B MeRIP-qPCR analysis of HBx mRNA, compared to the positive control. C MeRIP-qPCR analysis of HBx 3’UTR WT and MT mRNA. D RNA decay assay to detect the half-life of HBx 3’UTR WT and MT mRNA. HepG2 cells were transfected with HBx 3’UTR WT or MT plasmids and RT-qPCR was performed after actinomycin D was added. E MeRIP-qPCR analysis of HBx mRNA after silencing of ALKBH5. F Western blot analysis of HBx, WDR5 and H3K4me3 protein after silencing of ALKBH5. G Cartoon illustration of the positive feedback loop of HBx-ALKBH5.WT, widetype; MT, mutant