目的·探究黏蛋白1（mucin 1，MUC1）调控肿瘤细胞增殖、迁移和干性维持的功能位点。方法·通过癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库分析寻找MUC1基因在不同癌症中的突变特征，对不同MUC1突变位点进行分析及定位，并按突变出现频率排序；通过Western blotting筛选出突变频率较高且蛋白稳定表达的MUC1突变体，利用乳腺癌细胞株BT549敲除MUC1细胞系和乳腺非转化细胞株MCF-10A，应用慢病毒表达系统构建MUC1野生型（MUC1-WT）和突变体稳定表达的细胞系。采用免疫荧光法检测不同MUC1突变体的细胞定位。以MUC1-WT为阳性对照、MUC1-AQA功能丧失突变体为阴性对照，对不同突变细胞的肿瘤生物学功能进行分析：通过细胞计数试剂盒-8（cell counting kit-8，CCK-8）及克隆形成实验检测细胞增殖能力；通过划痕实验及Transwell实验检测细胞迁移能力；通过成球实验检测细胞干性。使用PyMOL软件分析MUC1突变体结构定位并通过蛋白质对接软件（ZDOCK Server）进行分子对接分析。结果·在TCGA数据库中得到102个位于MUC1编码区的突变，其中P418S、S251R、V359I、N271S、N465H 5个错义突变出现频率较高且位于非数目可变串联重复序列（non-variable number of tandem repeats，non-VNTR）区域。进一步检测发现MUC1-S251R、N271S、V359I突变体可稳定表达；细胞定位分析发现这3个突变体主要分布于细胞质，同时细胞核也有一定的分布，核质比与野生型未见明显差异。表达不同MUC1突变体细胞的肿瘤生物学功能分析发现：① MUC1-WT高表达显著增强BT549和MCF-10A细胞的增殖能力；与MUC1-WT细胞相比，MUC1-AQA、S251R、N271S突变体细胞增殖能力下降，但MUC1-V359I突变体细胞与MUC1-WT细胞具有相似的增殖能力。② MUC1-WT高表达细胞的迁移能力显著增强，而MUC1-AQA细胞迁移能力减弱。在BT549细胞中，MUC1-S251R与MUC1-V359I突变体细胞迁移能力与MUC1-WT细胞相似，但MUC1-N271S细胞的迁移能力较MUC1-WT细胞降低。在MCF-10A细胞中，MUC1-N271S与MUC1-V359I细胞的迁移能力接近MUC1-WT细胞；但MUC1-S251R细胞较MUC1-WT细胞迁移能力显著下降。③ MUC1-WT高表达显著增强2种细胞的干性，而MUC1-AQA细胞干性丧失；MUC1-N271S、V359I与MUC1-WT具有相似的维持细胞干性的能力，而MUC1-S251R使细胞干性减弱。PyMOL软件分析结果显示，MUC1-N271S及V359I位于海胆精子蛋白-肠激酶-聚集蛋白（sperm protein-enterokinase-agarin，SEA）区域及附近，分别处于loop区及β-折叠处；分子对接结果显示，MUC1-WT及V359I与表皮生长因子受体（epidermal growth factor receptor，EGFR）胞外域形成复合物的稳定性强于MUC1-N271S和S251R，其稳定性排序为V359I>WT>N271S>S251R。结论·MUC1突变体对肿瘤细胞的生物学功能具有不同影响，其对增殖能力影响可能与EGFR信号通路相关。MUC1-V359I与MUC1-WT相似，并未影响MUC1对肿瘤细胞增殖、迁移及干性维持的作用；而MUC1-S251、N271位点可能参与细胞增殖和迁移的信号通路调控且MUC1-S251位点对维持细胞干性较为重要。

Objective ·To identify the functional motifs of mucin 1 (MUC1) involved in regulating tumor cell proliferation, migration, and stemness maintenance. Methods ·Mutational characteristics of the MUC1 gene across different cancers were identified from The Cancer Genome Atlas (TCGA) database. Various MUC1 mutation sites were analyzed and localized, followed by ranking based on mutation frequency. Western blotting was used to screen high-frequency MUC1 mutants with stable protein expression. BT549 cell line with MUC1 knocked out and MCF-10A cell line were used to stably overexpress MUC1 wild-type (MUC1-WT) and mutants by using lentiviral technology. Immunofluorescence was used to detect the cellular localization of MUC1 mutants. Using MUC1-WT as a positive control and MUC1-AQA, a loss-of-function mutant, as a negative control, the biological functions of different MUC1 mutant cells were analyzed: cell proliferation ability was assessed by cell counting kit-8 (CCK-8) assay and colony formation assay; cell migration ability was evaluated by wound-healing and Transwell assays; cell stemness was examined by sphere formation assay. Structural localization of MUC1 mutants was analyzed by using PyMOL software, and molecular docking analysis was performed by using a protein docking software (ZDOCK Server). ·Results A total of 102 mutations located in the MUC1 coding region were identified in the TCGA database, among which five missense mutations (P418S, S251R, V359I, N271S, and N465H) exhibited higher frequencies and were located in the non-variable number of tandem repeats (non-VNTR) region. Further examination revealed that the MUC1-S251R, N271S, and V359I mutants could be stably expressed. The cellular localization assay indicated that these three mutants predominantly localized in the cytoplasm, but were also presented in the nucleus. The nuclear-to-cytoplasmic ratio showed minimal differences between MUC1-WT and the mutants. Analysis of the tumorigenic biological functions of the cells expressing different MUC1 mutants revealed that: ① High expression of MUC1-WT significantly enhanced the proliferation ability of both BT549 and MCF-10A cells; the proliferation of MUC1-AQA, S251R, and N271S mutant cells was decreased compared to MUC1-WT cells, while MUC1-V359I mutant cells exhibited a similar proliferative profile to MUC1-WT cells. ② The migration ability of MUC1-WT high-expressing cells was significantly enhanced, whereas MUC1-AQA cells demonstrated attenuated migration. In the BT549 cells, the migration ability of MUC1-S251R and V359I cells was similar to that of MUC1-WT cells, whereas MUC1-N271S cells showed reduced migration. In the MCF-10A cells, the migration ability of MUC1-N271S and MUC1-V359I cells was similar to that of MUC1-WT cells, whereas MUC1-S251R cells exhibited significantly decreased migration. ③ Stemness was enhanced in both cell types with high MUC1-WT expression, while MUC1-AQA cells lost stemness; the cells with MUC1-N271S, V359I and MUC1-WT showed comparable maintenance of stemness, whereas MUC1-S251R cells demonstrated compromised stemness. PyMOL software analysis unveiled that MUC1-N271S and V359I were located in or around the sperm protein-enterokinase-agarin (SEA) region, specifically in the loop region and the β-sheet, respectively. The molecular docking analysis revealed that the stability of the complex formed by MUC1-WT or V359I with the extracellular domain of epidermal growth factor receptor (EGFR) surpassed that of MUC1-N271S or S251R, indicating a stability hierarchy of V359I>WT>N271S>S251R. ·Conclusion MUC1 mutants exhibit diverse impacts on the biological functions of tumor cells, with their effects on proliferation correlating with the EGFR signaling pathway. MUC1-V359I is similar to MUC1-WT, indicating a negligible effect on tumor cell proliferation, migration, and stemness maintenance. Conversely, MUC1-S251 and N271 sites may be involved in the regulation of signaling pathways governing cell proliferation and migration and the MUC1-S251 site plays a critical role in maintaining cell stemness.