Silencing YTHDF2 Induces Apoptosis of Neuroblastoma Cells In a Cell Line-Dependent Manner via Regulating the Expression of DLK1.

Abstract

Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children. The complications caused by traditional chemoradiotherapy seriously affect the quality of life of patients with NB. In this study, NGP, KCNR, and SH-SY5Y (SY5Y) cell lines were used. Retinoic acid (RA, 5 µM) was used to treat NB cells for 48 h. siRNAs were used to silence the expression of DLK1 or YTHDF2. Cell confluence was analyzed using IncuCyte ZOOM to evaluate cell proliferation of NB cells. RT-qPCR and western blotting were performed to detect the expression of target molecules. Annexin V/PI staining and Caspase-Glo 3/7 assay were performed to detect cell apoptosis. RNA m6A quantification, MeRIP-qPCR, and RIP-qPCR were performed. Results showed that RA treatment decreased the expression of DLK1 and YTHDF2 in NB cells, and low expression of DLK1 was correlated with good prognosis of patients. Knockdown of the expression of DLK1 or YTHDF2 inhibited cell proliferation and induced apoptosis of SY5Y cells, but not NGP and KCNR cells. Furthermore, we found that there are m6A modification sites in DLK1 mRNA, and the expression of m6A modified DLK1 mRNA increased after RA treatment, and YTHDF2 regulates the expression level of DLK1, and the expression of YTHDF2-bound DLK1 mRNA decreased after RA treatment. These suggest that YTHDF2 may regulate the proliferation and apoptosis of NB cells in a cell line-dependent manner by binding to the m6A modification site of DLK1 mRNA to affect its expression, and YTHDF2 and DLK1 are potential therapeutic targets for patients with NB.