m6A-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating BMPR1A Transcription

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, N6-methyladenosine (m6A) has been reported to be involved in HCC initiation and progression. However, whether m6A has oncofetal characteristics remains unknown.PurposeHepatocellular 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, N6-methyladenosine (m6A) has been reported to be involved in HCC initiation and progression. However, whether m6A has oncofetal characteristics remains unknown.Gene expression in HCC tissues and cells was detected using qPCR. The level of m6A 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).MethodsGene expression in HCC tissues and cells was detected using qPCR. The level of m6A 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).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 m6A hypermethylation of NUTM2B-AS1. The m6A methylation levels of NUTM2B-AS1 exhibit oncofetal characteristics. m6A methylation upregulates NUTM2B-AS1 expression by increasing NUTM2B-AS1 transcript stability. m6A-methylated NUTM2B-AS1 promotes HCC cell proliferation and stemness via epigenetically activating BMPR1A expression. NUTM2B-AS1 specifically binds to BMPR1A promoter. m6A-methylated NUTM2B-AS1 is recognized by the m6A reader YTHDC2, wh