目的：探讨甲基转移酶样因子3（METTL3）在食管鳞状细胞癌（ESCC）组织和细胞中的表达水平及其对ESCC 细胞糖 酵解和增殖能力的影响和潜在的分子机制。方法：基于TCGA 数据库分析METTL3 在ESCC 细胞中的表达及可能的富集通路。 收集2021 年1 月至2021 年6 月间在北川医学院附属医院外科手术切除的34 例ESCC 组织及相应癌旁组织，采用免疫组化法验证 ESCC 组织中METTL3 的表达。采用CCK-8 法和平板克隆形成实验检测干扰METTL3 后ESCC 细胞增殖能力的变化，利用比色 法检测干扰METTL3 后ESCC 细胞总RNA 中m6A 的表达水平，采用甲基化RNA 免疫沉淀定量PCR（MeRIP-qPCR）检测METTL3 对葡萄糖转运蛋白4（GLUT4）基因mRNA 的m6A 修饰水平的影响，采用WB 和qPCR 等技术探索METTL3 参与ESCC 细胞糖酵解 的生物学机制。结果：METTL3 在ESCC 组织以及细胞中均呈高表达（均P<0.001）。干扰METTL3 表达后，ESCC 细胞的增殖能 力明显减弱、细胞内总RNA 的m6A 修饰水平显著降低（均P<0.001）。此外，干扰METTL3 可显著抑制KYSE150 和TE-1 细胞中 GLUT4 基因mRNA 的m6A 修饰水平（均P<0.01），并通过下调GLUT4 的表达抑制葡萄糖的摄取以及乳酸的释放（均P<0.01），最 终下调mTORC1 通路活性并抑制ESCC 细胞的增殖；在干扰METTL3 的ESCC 细胞同时联合运用mTORC1 通路抑制剂显示有协 同的抗癌作用。结论：METTL3 介导的m6A 修饰通过调控GLUT4-mTORC1 信号轴影响ESCC 细胞的糖酵解及增殖。

Objective: To investigate the expression of methyltransferase-like 3 (METTL3) in esophageal squamous cell carcinoma (ESCC) tissue and cells and its effect on the glycolysis proliferation of ESCC cells as well as the potential molecular mechanisms. Methods: Based on the TCGA database, the expression of METTL3 and its possible enrichment pathway in ESCC were elucidated. Thirty-four ESCC tissues and corresponding paracancerous tissues were collected from surgical resections at the Affiliated Hospital of Beichuan Medical College between January 2021 and June 2021, the expression of METTL3 in ESCC tissues was verified by immunohistochemistry. The CCK-8 and colony formation assay were used to evaluate the change in the proliferation ability of ESCC cells after METTL3 knockdown. The expression level of m6A in total RNA of ESCC cells after METTL3 knockdown was detected by colorimetric method. Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) was used to detect the effect of METTL3 on the m6A modification level of glucose transporter 4 (GLUT4) mRNA. The biological mechanisms of METTL3 in the glycolysis metabolism of ESCC were evaluated using WB and qPCR. Results: The expression level of METTL3 was significantly increased in ESCC tissues as well as cell lines (all P<0.001). After the knockdown of METTL3, the proliferation ability of ESCC cells was significantly reduced, and the m6A modification level of total RNA was significantly reduced (all P<0.001). In addition, knockdown of METTL3 significantly inhibited the m6A modification level of GLUT4 mRNA in KYSE150 and TE-1 cells (all P<0.01), inhibited glucose uptake and lactate release by down-regulating GLUT4 expression (all P<0.01), and finally down-regulated the activity of mTORC1 pathway and inhibited the proliferation of ESCC cells. Moreover, a synergetic effect was found in METTL3-depleted ESCC cells combined with mTORC1 pathway inhibitor. Conclusion: METTL3-mediated m6A modification promotes glycolysis and proliferation of ESCC cells by regulating the GLUT4-mTORC1 signaling axis.

四川省应用基础研究计划项目（No.2021YJ0202）