N(6)-Methyladenosine-Modified LEAWBIH Drives Hepatocellular Carcinoma Progression through Epigenetically Activating Wnt/β-Catenin Signaling

PURPOSE: N(6)-methyladenosine (m(6)A) modification plays an important role in regulating RNA maturation, stability, and translation. Thus, m(6)A modification is involved in various pathophysiological processes including hepatocellular carcinoma (HCC). However, the direct contribution of m(6)A 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 m(6)A-modified long non-coding RNA (lncRNA) and investigated the effects of m(6)A 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 m(6)A 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 m(6)A 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 m(6)A-modified transcript. m(6)A modification increased LEAWBIH transcript stability. The m(6)A modification level of LEAWBIH was increased in HCC, and a high m(6)A modification level of LEAWBIH predicted poor survival. LEAWBIH promotes HCC cell proliferation, migration, and invasion in an m(6)A modification-dependent manner. Mechanistic investigations revealed that m(6)A-modified LEAWBIH activated Wnt/β-catenin signaling. m(6)A-modified LEAWBIH binds to the m(6)A 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: m(6)A-modified LEAWBIH exerts oncogenic effects in HCC by epigenetically activating Wnt/β-catenin signaling, highlighting m(6)A-modified LEAWBIH as a promising therapeutic target for HCC.