N⁶-Methyladenosine-Modified LEAWBIH Drives Hepatocellular Carcinoma Progression through Epigenetically Activating Wnt/β-Catenin Signaling

Abstract

Purpose

N⁶-methyladenosine (m⁶A) modification plays an important role in regulating RNA maturation, stability, and translation. Thus, m⁶A modification is involved in various pathophysiological processes including hepatocellular carcinoma (HCC). However, the direct contribution of m⁶A modifications to RNA function in HCC remains unclear. Here, we identified LEAWBIH (long non-coding RNA epigenetically activating Wnt/β-catenin signalling in HCC) as an m⁶A-modified long non-coding RNA (lncRNA) and investigated the effects of m⁶A on the function of LEAWBIH in HCC.

Methods

Quantitative polymerase chain reaction was performed to measure the gene expression in tissues and cells. The level of m⁶A modification was detected using a methylated RNA immunoprecipitation assay and single-base elongation- and ligation-based qPCR amplification method. Cell proliferation was evaluated using the Glo cell viability and CCK-8 assays. Cell migration and invasion were evaluated using Transwell migration and invasion assays. The mechanisms of m⁶A modified LEAWBIH were investigated using chromatin isolation by RNA purification, chromatin immunoprecipitation, and dual-luciferase reporter assays.

Results

LEAWBIH was highly expressed and correlated with poor survival in HCC patients. LEAWBIH was identified as a m⁶A-modified transcript. m⁶A modification increased LEAWBIH transcript stability. The m⁶A modification level of LEAWBIH was increased in HCC, and a high m⁶A modification level of LEAWBIH predicted poor survival. LEAWBIH promotes HCC cell proliferation, migration, and invasion in an m⁶A modification-dependent manner. Mechanistic investigations revealed that m⁶A-modified LEAWBIH activated Wnt/β-catenin signaling. m⁶A-modified LEAWBIH binds to the m⁶A reader YTHDC1, which further interacts with and recruits H3K9me2 demethylase KDM3B to CTNNB1 promoter, leading to H3K9me2 demethylation and CTNNB1 transcription activation. Functional rescue assays showed that blocking Wnt/β-catenin signaling abolished the role of LEAWBIH in HCC.

Conclusion

m⁶A-modified LEAWBIH exerts oncogenic effects in HCC by epigenetically activating Wnt/β-catenin signaling, highlighting m⁶A-modified LEAWBIH as a promising therapeutic target for HCC.

Keywords:

• hepatocellular carcinoma
• N⁶-methyladenosine
• histone methylation
• Wnt/β-catenin signaling

Abbreviations

m⁶A, N⁶-methyladenosine; HCC, Hepatocellular carcinoma; lncRNA, long non-coding RNA; TCGA, The Cancer Genome Atlas; LIHC, Liver Hepatocellular Carcinoma; LEAWBIH, long non-coding RNA epigenetically activating Wnt/β-catenin signaling in HCC; FBS, fetal bovine serum; qPCR, quantitative polymerase chain reaction; cDNA, complementary DNA; MeRIP, methylated RNA immunoprecipitation; SELECT, single-base elongation- and ligation-based qPCR amplification method; NC, negative control; EdU,5-ethynyl-2’-deoxyuridine; ChIRP: Chromatin isolation by RNA purification; ChIP, Chromatin immunoprecipitation; GSEA, Gene Set Enrichment Analysis.

Ethics Approval and Consent to Participate

Human specimens were collected at the Affiliated Hospital of Youjiang Medical University for Nationalities. Written informed consent was obtained from all participants. This study was conducted in accordance with the Declaration of Helsinki and was reviewed and approved by the Affiliated Hospital of Youjiang Medical University for Nationalities Institutional Review Board.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This study was supported by the Guangxi Science and Technology Project (2021AC20006) and the High-Level Personnel Project of the Affiliated Hospital of Youjiang Medical University for Nationalities (R202210307).