[摘 要] 目的：探究蛋白酪氨酸磷酸酶D（PTPRD）去甲基化通过PI3K/Akt/mTOR通路对胃癌细胞增殖、迁移及化疗耐药性的影 响。方法：体外培养胃癌细胞MKN-74、MKN-45和人胃黏膜上皮细胞GES-1并检测PTPRD表达。常规培养MKN-45细胞及耐药 MKN-45/5-FU细胞，分别转染PTPRD空载体（NC组、NC/5-FU 组）、PTPRD 过表达腺病毒（PTPRD 组、PTPRD/5-FU组）、shRNA 空载体（sh-NC组、sh-NC/5-FU组）、shRNA-PTPRD慢病毒（sh-PTPRD组、sh-PTPRD/5-FU组）和PTPRD过表达腺病毒 + 10 μmol/L 740Y-P 处理（PTPRD + 740Y-P组、PTPRD + 740Y-P/5-FU组）。MTT法、划痕愈合实验检测各组细胞的增殖活力和迁移能力，细胞自噬实验 检测细胞的自噬水平，WB法检测细胞中EMT和PI3K/Akt/mTOR通路相关蛋白的表达。采用0、2.5、5、10、20、40 μmol/L的5-aza处 理MKN-45细胞，qPCR法、MTT法检测细胞中PTPRD mRNA表达和细胞增殖活力。结果：PTPRD mRNA和蛋白在胃癌细胞中均 呈低表达（P < 0.05）。与MKN-45组相比，PTPRD组自噬体与自噬溶酶体数量、PTPRD、上皮钙黏素（E-cadherin）、BAX蛋白表达均 增加（均P < 0.05），细胞增殖活力、细胞迁移率、p-PI3K、波形蛋白（vimentin）、p-Akt、p-mTOR蛋白表达均降低（均 P < 0.05）， sh-PTPRD 组细胞增殖活力、细胞迁移率、p-PI3K、vimentin、p-Akt、p-mTOR蛋白表达均增加（均P < 0.05），自噬体与自噬溶酶体数 量、PTPRD、E-cadherin、BAX蛋白表达均减少（均P < 0.05）；与PTPRD组相比，PTPRD + 740Y-P组细胞增殖活力、细胞迁移率、p-PI3K、 vimentin、p-Akt、p-mTOR 蛋白表达均增加（均 P < 0.05），自噬体与自噬溶酶体数量、PTPRD、E-cadherin、BAX蛋白表达减少（均 P < 0.05）。随着5-aza浓度的增加，MKN-45细胞中PTPRD mRNA表达增加、细胞增殖活力均降低（均P < 0.05）。与MKN-45/5-FU组 相比，PTPRD/5-FU组细胞迁移率、细胞增殖活力均降低（均P < 0.05），sh-PTPRD/5-FU组细胞迁移率、细胞增殖活力均增加（均P < 0.05）； 与PTPRD/5-FU组相比，PTPRD + 740Y-P/5-FU组细胞迁移率、细胞增殖活力均增加（均P < 0.05）。结论：PTPRD在胃癌细胞中呈 低表达状态，PTPRD去甲基化可能通过抑制PI3K/Akt/mTOR信号通路抑制胃癌细胞增殖、迁移并增强其对化疗的敏感性。

[Abstract] Objective: To investigate the effect of protein tyrosine phosphatase D (PTPRD) demethylation on the proliferation, migration, and chemoresistance of gastric cancer (GC) cells through the phosphatidyl inositol 3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Methods: The gastric cancer MKN-74 and MKN-45 cells, as well as human gastric mucosal epithelial GES-1 cells, GES-1 were cultured in vitro and PTPRD expression was detected. MKN-45 cells and their drug-resistant variant MKN-45/ 5-FU cells were routinely cultured and transfected with various vectors: PTPRD empty vector (NC group, NC/5-FU group), PTPRD overexpressing adenovirus (PTPRD group, PTPRD/5-FU group), shRNA empty vector (sh-NC group, sh-NC/5-FU group), shRNA PTPRD lentivirus (sh-PTPRD group, sh-PTPRD/5-FU group), and PTPRD overexpressing adenovirus + 10 μmol/L 740Y-P treatment (PTPRD + 740Y-P group, PTPRD + 740Y-P/5-FU group). MTT assay and wound healing assay were used to assess cell proliferation and migration. Cell autophagy levels were assessed using autophagy assay, and the expression of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR pathway related proteins was detected using western blot (WB). MKN-45 cells were treated with 0, 2.5, 5, 10, 20 and 40 μmol/L 5-aza solutions, and the PTPRD mRNA expression and cell proliferation in MKN-45 cells were detected using qPCR and MTT assays. Results: PTPRD mRNA and protein were significantly downregulated in gastric cancer cells (P < 0.05). Compared with the MKN-45 group, the numbers of autophagosomes and autophagosomes, as well as the protein expression of PTPRD, E-cadherin, and BAX significantly increased in the PTPRD group (all P < 0.05), while cell proliferation, migration rate, and protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR decreased significantly (all P < 0.05); However, in the sh-PTPRD group, cell proliferation activity, migration rate, and protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR increased notably, while the quantity of autophagosomes, autophagosomes, and protein expression of PTPRD, E-cadherin, and BAX decreased (all P < 0.05). Compared with the PTPRD group, the PTPRD + 740Y-P group showed an increase in cell proliferation activity, migration rate, protein expression of p-PI3K, vimentin, p-Akt, and p-mTOR (all P < 0.05), and a decrease in number of autophagosomes, autophagosomes, and protein expression of PTPRD, E-cadherin, and BAX (all P < 0.05). With the increase of 5-aza concentration, the mRNA expression of PTPRD in MKN-45 cells increased (P < 0.05), while the cell proliferation activity decreased (P < 0.05). Compared with the MKN-45/5-FU group, the cell migration rate and proliferation activity decreased in PTPRD/5-FU group, while the sh-PTPRD/5-FU group showed an increase in cell migration rate and proliferation activity (all P < 0.05). Compared with the PTPRD/5-FU group, the PTPRD + 740Y-P/5-FU group showed an increase in cell migration rate and proliferation activity (all P < 0.05). Conclusion: PTPRD is downregulated in GC cells, and its demethylation may inhibit proliferation and migration of GC cells and enhance chemosensitivity by suppressing the PI3K/Akt/mTOR pathway.

内蒙古自治区科技计划项目（No. 2020GG0171）；内蒙古自治区高等学校科学研究项目（No. NJZZ23013）