RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner

Background N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). Methods The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. Results ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. Conclusion ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

背景 N6 - 甲基腺苷（m6A）是真核生物mRNA最丰富的可逆甲基化修饰，在肿瘤发生中起着至关重要的作用。本研究旨在探讨m6A去甲基化酶ALKBH5在胰腺癌（PC）中的作用。 方法 在胰腺癌队列中评估ALKBH5的表达及其临床病理影响。通过功能获得和功能缺失分析研究ALKBH5对胰腺癌细胞生物学特性的影响。皮下和原位模型进一步揭示了ALKBH5在肿瘤生长中的作用。进行mRNA和m6A测序以及m6A甲基化RNA免疫沉淀 - qPCR（MeRIP - qPCR）实验以确定ALKBH5对PER1的靶向作用。通过染色质免疫沉淀（ChIP）和荧光素酶实验研究ALKBH5启动子中的P53结合位点，以揭示ALKBH5与PER1激活的ATM - CHK2 - P53/CDC25C信号通路之间的相互作用。 结果 ALKBH5缺失是胰腺癌患者发病及不良临床病理表现的特征。ALKBH5过表达在体外降低了肿瘤的增殖、迁移和侵袭活性，在体内改善了肿瘤生长，而ALKBH5敲低则促进了胰腺癌进展。从机制上讲，ALKBH5以m6A - YTHDF2依赖的方式通过m6A去甲基化在转录后激活PER1。PER1上调导致ATM - CHK2 - P53/CDC25C信号通路重新激活，从而抑制细胞生长。P53诱导的ALKBH5转录激活作为一个反馈回路调节胰腺癌中的m6A修饰。 结论 ALKBH5通过消除m6A调节PER1的转录后激活而作为胰腺癌的抑制因子，这可能为胰腺癌的诊断和治疗突出一种基于去甲基化的方法。