Integrated analysis based on RNA immunoprecipitation sequencing (RIP-seq) and m5C methylated RNA immunoprecipitation and sequencing (MeRIP-seq) assays to identify the target genes of NSUN2/YBX1-m5C axis in ESCC progression

The modification and recognition of 5-methylcytosine (m5C) are involved in the initiation and progression of various tumor types. However, the precise role and potential mechanism of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we found that YBX1 was frequently upregulated in ESCC compared with matched nontumor tissues. Gain- and loss-of-function assays showed that YBX1 promoted the proliferation and metastasis of ESCC cells both in vitro and in vivo. Functional studies revealed that NOP2/Sun RNA methyltransferase family member 2 (NSUN2) is a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, integrated analysis based on RNA immunoprecipitation sequencing (RIP-seq) and m5C methylated RNA immunoprecipitation and sequencing (MeRIP-seq) assays identified spermine oxidase (SMOX) as a target gene containing an m5C site in its coding sequence (CDS) region, which coincided well with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restored the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Moreover, YBX1 activated the mTORC1 signaling pathway by stabilizing SMOX mRNA. Our study revealed that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, thus providing a valuable therapeutic target for ESCC. Overall design: We performed an m5C meRIP-seq assay to investigate the effect of silencing NSUN2 on the m5C profiles of ESCC cells RNA immunoprecipitation sequencing (RIP-seq) was performed to identify the downstream transcripts of YBX1 with m5C sites in ESCC Overlaying YBX1-binding targets (Fold enrichment >10) with transcripts displaying reduced m5C sites (Foldchange >200) and expression level (logFC <-2) following NSUN2 knockdown in KYSE150 cells.