m6A-lncRNA landscape highlights reduced levels of m6A modification in glioblastoma as compared to low-grade glioma.

Background: Efforts to understand the interplay between m6A (N6-methyladenosine) modification and long non-coding RNAs (lncRNAs) in the pathogenesis of various diseases, including cancer, have recently attracted considerable attention.

Methods: Herein, we profiled epitranscriptome-wide m6A modifications within lncRNAs at single m6A site resolution across different grades of gliomas (Glioblastomas (GB): n = 17, Low grade gliomas (LGG): n = 9) using direct RNA long-read sequencing.

Results: Our analysis demonstrated that, 1) 98.5% of m6A-modified RRACH motifs were present within mRNA transcripts, while only 1.16% were conspicuous within lncRNAs. Importantly, LGGs exhibited a higher m6A abundance (23.73%) compared to the GB transcriptome (15.84%). 2) The m6A profiles of lncRNAs differed significantly between gliomas, with unsupervised cluster analysis revealing two clusters (C1, C2). LGG dispersed between C1 and C2 clusters while GB stayed mainly in C1. Clinical feature association analysis between m6A clusters showed the tendency of m6A to be associated with higher malignancy grade (p = 0.053), while significant association was observed with higher Ki-67 proliferation index (p = 0.04), and tumor location (p < 0.01). Specifically, brain tumors located in cerebellum (n = 3) were highly m6A modified on lncRNAs as compared to tumors in other locations (frontal lobe, n = 5, p = 0.003; frontotemporal lobe, n = 2, p = 0.08; occipital, n = 2, p = 0.038; parietal, n = 2, p = 0.007; temporal, n = 11, p < 0.001). Cox regression analysis showed that the status of lncRNAs m6A modifications had no significant value in predicting post-surgical survival time in our GB or LGG cohorts. The trend of higher lncRNA expression in m6A methylated group was observed for the majority of lncRNAs, while only MIR9-1HG (r = 0.439, p = 0.028) and ZFAS1 (r = 0.609, p < 0.05) m6A showed statistically significant positive correlations in gliomas. A high-resolution m6A study revealed that mRNA levels of m6A writers and erasers in gliomas do not reflect global m6A methylation.

Conclusions: Overall, we provide evidence that m6A lncRNAs are strongly modulated in gliomas, representing biologically distinct subgroups. Ten novel differentially methylated lncRNAs were identified in gliomas, which might exert regulatory role in glioma cells. These findings may provide a basis for further deeper research on the role of m6A lncRNAs in gliomas.