[摘 要] 目的：探讨烯醇化酶1（ENO1）对胃癌细胞增殖、迁移及侵袭的影响及其分子机制。方法：通过WB法检测ENO1在 人胃癌细胞HGC27、MKN-45、N-87、MGC803、BGC-823及人胃黏膜上皮细胞GES-1中的表达水平，利用CRISPR、过表达等基因 编辑工具分别构建ENO1敲低及敲低—恢复表达细胞株，将MKN-45和BGC-823细胞分别分组为Ctrl组、ENO1 KD组、ENO1 KD-OE组。通过克隆形成实验、EdU染色、划痕实验、Transwell实验、流式细胞术检测敲低或敲低—恢复表达ENO1对胃癌细胞 增殖、迁移、侵袭及凋亡的影响。构建胃癌细胞裸鼠移植瘤模型，通过小动物活体成像技术及肿瘤组织块测量观察ENO1对肿瘤 生长的影响。在MKN-45细胞中通过RNA干扰技术沉默ENO1，利用RNA免疫共沉淀-转录组测序（RIP-Seq）结合生物信息学分 析技术鉴定ENO1下游的靶基因，分析ENO1调控胃癌细胞增殖、迁移及侵袭的分子机制。结果：ENO1在胃癌细胞中表达水平 均显著升高（P < 0.01或P < 0.001）。在MKN-45和BGC-823细胞中敲低ENO1可显著降低胃癌细胞的增殖、迁移、侵袭能力并促 进细胞凋亡（P < 0.001或P < 0.000 1）；回复实验结果显示，恢复ENO1的表达后，胃癌细胞的增殖、迁移、侵袭能力均显著提高并 减少细胞凋亡（P < 0.05或P < 0.01或P < 0.001或P < 0.000 1）。裸鼠荷瘤实验结果表明，敲低ENO1的表达可以显著抑制裸鼠移 植瘤的生长（P < 0.000 1）。与ENO1蛋白具有相互作用的差异基因主要富集于RNA剪切相关通路，且ENO1蛋白与PKM基因具 有相互作用，胃癌组织中ENO1与PKM2基因表达事呈正相关（r = 0.886）。结论：ENO1在胃癌细胞中呈高表达，其通过与PKM 的前体mRNA相互作用影响PKM的RNA剪接过程，进而调控PKM2的表达水平并促进胃癌的进展。

[Abstract] Objective: To investigate the effects of enolase 1 (ENO1) on the proliferation, migration, and invasion of gastric cancer cells and its underlying molecular mechanisms. Methods: The 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. Results: ENO1 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). Conclusion: ENO1 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.

[基金项目] 甘肃省自然科学基金（No. 24JRRA337，No. 24JRRA364）