mA-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating Transcription.

Purpose: Hepatocellular carcinoma (HCC) is one of the most lethal malignancies in the world. Oncofetal proteins are the optimal diagnostic biomarkers and therapeutic targets for HCC. As the most abundant modification in RNA, N-methyladenosine (mA) has been reported to be involved in HCC initiation and progression. However, whether mA has oncofetal characteristics remains unknown.

Methods: Gene expression in HCC tissues and cells was detected using qPCR. The level of mA methylation was determined using methylated RNA immunoprecipitation assay. The biological roles of NUTM2B-AS1 in HCC were detected using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, and spheroid formation assays. The mechanisms underlying the roles of NUTM2B-AS1 were explored using RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and assay for transposase-accessible chromatin (ATAC).

Results: NUTM2B-AS1 was identified as a novel oncofetal long noncoding RNA that was upregulated in the fetal liver and HCC and silenced in adult liver tissues. METTL3 and METTL16 induce mA hypermethylation of NUTM2B-AS1. The mA methylation levels of NUTM2B-AS1 exhibit oncofetal characteristics. mA methylation upregulates NUTM2B-AS1 expression by increasing NUTM2B-AS1 transcript stability. mA-methylated NUTM2B-AS1 promotes HCC cell proliferation and stemness via epigenetically activating expression. NUTM2B-AS1 specifically binds to promoter. mA-methylated NUTM2B-AS1 is recognized by the mA reader YTHDC2, which further binds to the H3K4 methyltransferase MLL1. mA-methylated NUTM2B-AS1 recruits YTHDC2 and MLL1 to promoter, leading to increased H3K4me3 and chromatin accessibility at promoter. Functional rescue assays suggest that BMPR1A is a critical mediator of the oncogenic role of mA-methylated NUTM2B-AS1 in HCC.

Conclusion: METTL3- and METTL16-mediated mA methylation of NUTM2B-AS1 is a novel oncofetal molecular event in HCC that promotes HCC stemness via epigenetically activating transcription.