Analysis of M6A Modification Level at RRACH Motifs in LINC00461, GAS5, and NEAT1 Genes in Gliomas

Abstract

Glioblastoma (GB) is the most common and aggressive brain tumor, characterized by high resistance to therapy and by inter- and intra-tumoral heterogeneity [1]. Epitranscriptomic modifications may modulate key genes involved in glioblastoma cell metabolism and contribute to their pathogenesis by increasing their heterogeneity, plasticity, and malignancy [2]. M6A RNA methylation is a dynamic and reversible modification process which is highly involved in the initiation and development of numerous malignancies [3]. Recent studies propose that the m6A RNA methylation process offers potential targets for cancer therapy in the future. Snap-frozen tumor tissue from 16 GB and 8 LGG patients were used for isolation of total RNA with TRIzol Reagent. Poly-A enriched RNA was used for direct-RNA sequencing by Oxford Nanopore Technologies. The “nf-core/nanoseq” pipeline (ver.:2.0.1) was used for detection of RNA modifications. First, according to the literature in our RNA-seq data we selected three genes (LINC00461, GAS5 and NEAT1) which are involved in glioma progression, regulation of tumor cell growth, invasion, migration and metastasis. Second, our data analysis showed that m6A marks were lower in glioblastoma at gene level in all selected lncRNAs. There were significant differences between survival dependency and m6A modification level in LINC00461. Next, the most frequently modified RRACH motif in tumor samples was found to be AAACT in the LINC00461, GAS, and NEAT1 genes. AAACT motif was found to be mostly modified at the positions 3316 and 3892 of LINC00461 gene transcript 3-exon in GB and LGG samples, with a higher modification detected in GB. While in GAS5 the most modified positions were 513 and 604 in the 11-exon in both GB and LGG, being higher in LGG. NEAT1 gene at the positions 3184 and 3723 of AAACT motif were identified having a higher level of m6A in LGG, whereas were unmodified in GB samples at the 3184 position. Global m6A modification level was higher in LGG samples, as compared to higher malignancy grade glioblastomas. The most frequently modified RRACH motif in analyzed genes was identified AAACT and modification level differed depending on location in the gene. The link between m6A modification and lncRNAs offers a new perspective and suggests that m6A modification and lncRNAs may be important prognostic markers and therapeutic targets for gliomas.