N⁶-Methyladenosine-Modified LEAWBIH Drives Hepatocellular Carcinoma Progression through Epigenetically Activating...

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.

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.

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.

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.