ObjectiveTo investigate the effects of enolase 1 (ENO1) on the proliferation, migration, and invasion of gastric cancer cells and its underlying molecular mechanisms.MethodsThe expression levels of ENO1 in human gastric cancer cell lines (HGC27, MKN-45, N-87, MGC803, BGC-823) and human gastric mucosal epithelial cells (GES-1) were detected using WB assay. Gene editing tools such as CRISPR and overexpression system were used to construct ENO1 knockdown and knockdown-rescue cell lines. Both MKN-45 and BGC-823 cells were grouped into control (Ctrl) group, ENO1 knockdown (ENO1 KD) group, and ENO1 knockdown-rescue (ENO1 KD-OE) group. The effects of ENO1 knockdown or ENO1 knockdown-rescue on the proliferation, migration, invasion, and apoptosis of gastric cancer cells were evaluated using colony formation assay, EdU staining, scratch wound healing assay, Transwell chamber assay and flow cytometry. Additionally, a xenograft model was established in nude mice, and the effects of ENO1 on tumor growth were monitored using small animal in vivo imaging and tumor tissue block measurement. ENO1 was silenced in MKN-45 cells employing RNA interference technology, and the downstream target genes of ENO1 were identified using RNA co-immunoprecipitation sequencing (RIP-seq) and bioinformatics analysis. The molecular mechanisms by which ENO1 regulates the proliferation, migration and invasion of gastric cancer cells was also analyzed.ResultsENO1 was significantly upregulated in gastric cancer cell lines (P < 0.01 or P < 0.001). ENO1 knockdown significantly inhibited proliferation, migration, and invasion while promoting apoptosis in MKN-45 and BGC-823 cells (P < 0.001, P < 0.000 1). Rescue experiments showed that restoring ENO1 expression significantly enhanced cell proliferation, migration, invasion, and inhibited apoptosis (P < 0.05, P < 0.01, P < 0.001, P < 0.000 1). In vivo experiments demonstrated that ENO1 knockdown significantly inhibited tumor growth in nude mice (P < 0.000 1). The differentially expressed genes interacting with ENO1 protein were primarily enriched in pathways related to RNA splicing. Additionally, ENO1 protein was found to interact with the PKM gene, and their expressions showed a positive correlation in gastric cancer tissues (r = 0.886).ConclusionENO1 is highly expressed in gastric cancer cells. ENO1 interacts with precursor mRNA of PKM to influence its RNA splicing process, thereby regulating PKM2 expression and promoting gastric cancer progression.