目的：探讨细胞色素c氧化酶铜伴侣蛋白17（COX17）是否参与去甲泽拉木醛（DML）抑制胃癌细胞增殖和侵袭的作用。方法： 采用5、10和20 μmol/L DML处理MGC-803细胞，通过显微镜计数和Transwell实验评价DML对细胞增殖和侵袭的影响；采用标记定量蛋白质组学比较DML处理组与DMSO组的蛋白表达谱，结合差异表达分析和GO功能富集筛选候选蛋白，并采用Western blotting验证DML处理后COX17蛋白表达变化。分别构建COX17敲低和过表达稳定细胞株，通过细胞计数和Transwell实验评价COX17对MGC-803细胞增殖和侵袭的影响。采用分子对接和50 ns分子动力学模拟分析DML与COX17的潜在结合模式及复合物构象稳定性；进一步通过COX17过表达回复实验评价COX17在DML抑制细胞增殖和侵袭过程中的作用。结果：DML显著抑制MGC-803细胞增殖和侵袭，且随DML浓度升高，抑制作用逐渐增强（均P < 0.000 1）。蛋白质组学共筛选出72个差异表达蛋白，其中21个上调、51个下调；结合差异表达特征、GO功能富集结果及前期研究基础，选取COX17进行后续验证。DML可显著下调MGC-803细胞中COX17蛋白表达（P < 0.000 1）。敲低COX17可抑制细胞增殖和侵袭，过表达COX17则促进上述表型。分子对接提示DML与COX17之间可能存在疏水接触、氢键和盐桥等相互作用，对接评分为?6.8 kcal/mol；分子动力学模拟显示DML-COX17复合物在50 ns模拟过程中整体趋于相对稳定。COX17过表达可部分减弱DML对MGC-803细胞增殖和侵袭的抑制作用（P < 0.01）。结论：DML可抑制MGC-803细胞增殖和侵袭并下调COX17蛋白表达，COX17可能参与DML对胃癌细胞恶性表型的调控。

Objective: To investigate whether cytochrome c oxidase copper chaperone 17 (COX17) is involved in the inhibitory effects of demethylzeylasteral (DML) on the proliferation and invasion of gastric cancer cells. Methods: MGC-803 cells were treated with DML at 5, 10 and 20 μmol/L, and the effects of DML on cell proliferation and invasion were evaluated by manual cell counting and Transwell assay, respectively. Label-free quantitative proteomics was applied to compare the protein expression profiles between the DML-treated group and the DMSO group, and candidate proteins were screened by differential expression analysis combined with Gene Ontology (GO) functional enrichment analysis. The change in COX17 protein expression after DML treatment was verified by Western blotting. COX17-knockdown and COX17-overexpressing stable cell lines were constructed separately, and the effects of COX17 on the proliferation and invasion of MGC-803 cells were evaluated by cell counting and Transwell assay. Molecular docking and 50 ns molecular dynamics simulation were used to analyze the potential binding mode between DML and COX17 as well as the conformational stability of the complex. The role of COX17 in the DML-induced inhibition of cell proliferation and invasion was further evaluated by a COX17 overexpression rescue experiment. Results: DML significantly inhibited the proliferation and invasion of MGC-803 cells, and the inhibitory effect was progressively enhanced with increasing DML concentration (all P < 0.000 1). A total of 72 differentially expressed proteins were screened by proteomics, of which 21 were upregulated and 51 were downregulated. Based on these differential expression characteristics, the GO enrichment results and our previous findings, COX17 was selected for further validation. DML significantly downregulated COX17 protein expression in MGC-803 cells (P < 0.000 1). Knockdown of COX17 inhibited cell proliferation and invasion, whereas overexpression of COX17 promoted these malignant phenotypes. Molecular docking suggested that interactions such as hydrophobic contacts, hydrogen bonds and salt bridges might exist between DML and COX17, with a docking score of ?6.8 kcal/mol. Molecular dynamics simulation showed that the DML-COX17 complex tended to be relatively stable overall during the 50 ns simulation. Overexpression of COX17 partially attenuated the inhibitory effects of DML on the proliferation and invasion of MGC-803 cells (P < 0.01). Conclusion: DML can inhibit the proliferation and invasion of MGC-803 cells and downregulate COX17 protein expression, and COX17 may be involved in the regulation of the malignant phenotypes of gastric cancer cells by DML.

贵州省高层次创新型人才“千层次”人才培养项目（GZSYQCC[2020]009）