Abstract:

This study aimed to investigate the effect and mechanism of the N6-methyladenosine （m⁶A） demethylase alkB homolog 5 （ALKBH5） on cisplatin （DDP） chemosensitivity in non-small cell lung cancer （NSCLC） by regulating the m⁶A methylation of CPNE1 （Copine 1） mRNA. The expression levels of ALKBH5 and CPNE1 in lung cancer cell lines were detected by qRT-PCR and Western blot. The sequence-based RNA adenosine methylation site predictor （SRAMP） was used to predict the m⁶A methylation sites of CPNE1， Me-RIP assay was performed to detect its methylation level， and RNA immunoprecipitation （RIP） assay was conducted to verify the binding between ALKBH5 and CPNE1 mRNA. Lung cancer H157 cells were transfected with OE-CPNE1， si-CPNE1， and si-ALKBH5， respectively. Cell viability was detected by CCK-8 assay to calculate the IC₅₀ value， colony formation assay was used to evaluate cell proliferation， and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） staining was employed to detect cell apoptosis. The PI3K/AKT activator 740Y-P was added to si-CPNE1-transfected cells to analyze the role of CPNE1 in the PI3K/AKT pathway. The results showed that the expressions of ALKBH5 and CPNE1 in NSCLC cells were significantly higher than those in normal lung epithelial cells （P<0.05）. Knockdown of ALKBH5 decreased the expression of CPNE1 and increased its m⁶A methylation level （P<0.05）. The RIP assay confirmed a direct binding between ALKBH5 and CPNE1 mRNA. Inhibition of ALKBH5 or CPNE1 significantly enhanced the cytotoxic effect of cisplatin on lung cancer cells， which was manifested by decreased cell viability， inhibited proliferation， and increased apoptosis （P<0.05）. In addition， the PI3K/AKT activator 740Y-P could rescue the pathway inhibition and chemoresistance phenotype induced by CPNE1 knockdown. These findings indicated that ALKBH5 upregulated CPNE1 expression in an m⁶A-dependent manner and activated the PI3K/AKT pathway， thereby reducing the cisplatin chemosensitivity of NSCLC cells.

Key words: non-small cell lung cancer, m⁶A methylation, ALKBH5, CPNE1, P13/AKT, chemosensitivity