GLIOBLASTOMAS ARE CHARACTERIZED BY A REDUCED NUMBER OF M6A SITES IN LNCRNAs

Recent studies have suggested the potential role of posttranscriptional N6-methyladenosine (m6A) RNA modifications in diverse biological processes, including tumor development. In contrast to intensively studied mRNA modifications, insufficient information is known so far about the epitranscriptome of long non-coding RNAs (lncRNAs). Furthermore, an epitranscriptome of LncRNAs in glioma pathogenesis has not been previously presented. Thus, this study aimed to profile m6A modifications within lncRNAs in different grades of gliomas using direct RNA long-read sequencing, with the goal of yielding both nucleotide and transcript-resolved m6A hits as biomarkers for tumor progression. Here we explored ONT direct RNA-seq to detect N6-methyladenosine (m6A) sites within lncRNAs' RRACH motifs in 26 glioma tumor tissues. Generally, 98.5% of m6A-modified RRACH motifs were detected in mRNA transcripts, and 1.16% in lncRNAs. We identified 60-748 unique m6A-modified lncRNAs for individual gliomas. On average, 15.84% of all RRACH motifs within lncRNAs were modified in glioblastomas (GB), while the m6A frequency reached 23.73% in the low-grade glioma (LGG) group. Unsupervised clustering analysis based on lncRNAs' m6A status resulted in three m6A clusters. Patients within the highly modified lncRNAs cluster (C3) experienced significantly longer survival compared to patients with lower methylation rates who were clustered into C1/C2 (2901 days in C3 vs 439 days in C1, log-rank test p=0.019).  In summary, we discovered that lncRNAs are highly modified in LGG while multiple epi-marks, found in low-grade gliomas, are absent in GB tissues revealing m6A contribution to glioma pathology. Post-surgical tumor samples (17 glioblastoma (GB) and 9 diffuse astrocytoma (LGG)) sequenced using direct RNA sequecning approach for the detection of RNA expression and m6A modifications. Differences of RNA expression and m6A modifications were compared between GB and LGG groups.

近年研究表明，转录后N6-甲基腺苷（N6-methyladenosine，m6A）RNA修饰在包括肿瘤发生在内的多种生物学过程中发挥潜在作用。
与被广泛研究的信使RNA（mRNA）修饰不同，目前学界对长链非编码RNA（long non-coding RNAs，lncRNAs）的表观转录组（epitranscriptome）认知仍较为匮乏。
此外，目前尚无关于胶质瘤发病过程中lncRNAs表观转录组的相关研究报道。
因此，本研究旨在通过直接RNA长读长测序技术，对不同级别胶质瘤组织中lncRNAs的m6A修饰情况进行全景分析，以期获得兼具核苷酸与转录本分辨率的m6A位点，作为肿瘤进展的生物标志物。
本研究采用ONT直接RNA测序技术，对26例胶质瘤组织中lncRNAs的RRACH基序内的N6-甲基腺苷（m6A）位点进行检测。
整体而言，98.5%的携带m6A修饰的RRACH基序位于mRNA转录本中，仅1.16%存在于lncRNAs中。
本研究在每例胶质瘤样本中均鉴定出60~748个独特的携带m6A修饰的lncRNAs。
平均而言，胶质母细胞瘤（glioblastomas，GB）组中lncRNAs内所有RRACH基序的修饰比例为15.84%，而低级别胶质瘤（low-grade glioma，LGG）组的m6A修饰频率可达23.73%。
基于lncRNAs的m6A修饰状态进行无监督聚类分析，可将样本划分为3个m6A聚类簇。
高lncRNA修饰簇（C3）中的患者生存期显著长于被归入C1/C2簇的低甲基化水平患者（C3组生存期2901天，C1组为439天，对数秩检验p=0.019）。
综上，本研究发现LGG组中lncRNAs存在高度m6A修饰，而GB组织中缺失低级别胶质瘤中存在的多种表观标记，提示m6A修饰在胶质瘤病理进程中发挥重要作用。
本研究的样本为术后肿瘤组织，共17例胶质母细胞瘤（GB）与9例弥漫性星形细胞瘤（LGG），通过直接RNA测序技术检测其RNA表达水平与m6A修饰情况。
研究比较了GB与LGG组之间的RNA表达与m6A修饰差异。