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

Abstract

Huamei Wei,1,* Lizheng Huang,2,* Qi Lu,2,* Zheng Huang,2 Yanyan Huang,2 Zuoming Xu,3 Wenchuan Li,3 Jian Pu3,4 1Department of Pathology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, People’s Republic of China; 2Graduate College of Youjiang Medical University for Nationalities, Baise, People’s Republic of China; 3Department of Hepatobiliary Surgery, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, People’s Republic of China; 4Guangxi Clinical Medical Research Center of Hepatobiliary Disease, Baise, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jian Pu, Department of Hepatobiliary Surgery, Affiliated Hospital of Youjiang Medical University for Nationalities, No. 18 Zhongshan Two Road, Baise, 533000, People’s Republic of China, Email [email protected]: 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

Keywords

• hepatocellular carcinoma
• n6-methyladenosine
• histone methylation
• wnt/β-catenin signaling