| Aims: N6-methyladenosine(m6A)is the most abundant reversible methylation modification of eukaryotic m RNA.This study aimed to investigate the role and its specific regulatory mechanism of m6A demethylase ALKBH5 in pancreatic cancer(PC),and to find a new diagnosis and treatment strategy for PC.Methods: The protein expressing level of ALKBH5 in 42 pairs of PC patients was firstly detected by immunohistochemical,and analysis was carried out to detected the relationship between ALKBH5 protein expressing level and clinical stages,pathological grade and disease prognosis.Subsequently,gain-of-function and loss-of-function experiments were designed to explore the effects of ALKBH5 on the proliferation and invasion ability of PC cells in vitro respectively by CCK-8,plate cloning,EDU staining,cell scratching,and transwell experiments.Subcutaneous tumor models in nude mice further reveal the role of ALKBH5 in the growth of PC in vivo.The next-generation m RNA sequencing and Me RIP-seq technology were performed to analyze the transcriptome expression profile of ALKBH5 over-expressing PC cells,and then we intersected those genes up-regulated in m RNA expression profile sequencing and genes in the specific peak of the ALKBH5 untreated group in Me RIP-seq to find the potential target genes and signaling pathways of ALKBH5.WB,q RT-PCR,and Coip experiments were used to detected the effect of ALKBH5 on the Per1 /ATM/CHK2 pathway.Rescue experiments were designed to investigate the effects of Per1 on the proliferation,invasion and metastasis ability of PC.The dual luciferase expression vector containing ALKBH5 promoter was constructed and the TP53 binding site in ALKBH5 promoter was verified by Chip experiment.Results: The m6A level was increased and the expression level of ALKBH5 was decreased in PC tissues,and the expression level of ALKBH5 was significantly correlated with tumor invasiveness indicators such as pathological grade,clinical stages,and vascular invasion.Survival analysis results showed that low expression of ALKBH5 indicates a poor prognosis for PC patients.RNA-seq analysis detected an anti-proliferative and pro-apoptotic profile after ALKBH5 overexpression in Bx PC-3cells.Systematic GO and pathway analyses revealed that the upregulated genes were mostly enriched in cell cycle arrest-and apoptotic-related signaling;whereas the cell division signaling was suppressed.In vitro experiments revealed that ALKBH5 upregulation significantly inhibited the proliferation,migration and invasion ability of PC cells,and induced G2/M cell cycle arrest;whereas ALKBH5 knockdown led to the opposite effect.Subcutaneous transplantation experiments in nude mice also confirmed that ALKBH5 inhibit the proliferation and invasion ability of PC in vivo.Mechanistically,ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner.PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25 C signaling,which inhibited cell growth.P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC.ALKBH5 gene promoter held two TP53 binding site in the region of "-47/-64" and "-398/-412" and then activated ALKBH5 expression to form the ALKBH5-m6A-Per1-TP53-ALKBH5 positive feedback regulatory loop Conclusions: m6A modification is closely involved in human PC tumorigenesis.The m6A demethylase ALKBH5 serves as an anti-oncogene by inhibiting PC cell proliferation,migration and invasion,and promoting G2/M cell cycle arrest.ALKBH5 expression correlated with clinicopathological features and overall survival outcomes,and represented a predictive and progostic factor for patients with PC. |
PDF Full Text Request