目的：探究β-半乳糖苷α2,6-唾液酸转移酶1（ST6GAL1）和双特异性磷酸酶26（DUSP26）在结直肠癌（CRC）中的表达及其对巨噬细胞极化和肿瘤进展的影响。方法：选取CRC 患者126 例，采用免疫组织化学染色法检测肿瘤组织和癌旁组织中ST6GAL1、DUSP26 蛋白表达。采用Western blotting、免疫共沉淀、外泌体鉴定、流式细胞术、ELISA、免疫荧光等方法，分析ST6GAL1、DUSP26 及其相关信号通路对CRC 细胞外泌体介导的M2 型巨噬细胞极化、线粒体功能和内质网应激的影响。CRISPR/Cas9 和慢病毒系统构建ST6GAL1 敲除（ST6GAL1-KO）和DUSP26 过表达（DUSP26-OE）细胞模型，并采用小鼠肿瘤模型评估ST6GAL1-KO + DUSP26-OE联合干预对肿瘤生长、细胞增殖及凋亡的影响。结果：CRC患者肿瘤组织中ST6GAL1蛋白阳性率显著高于癌旁正常组织，而DUSP26蛋白阳性率显著低于癌旁正常组织（均P < 0.001）；CRC细胞中ST6GAL1蛋白表达显著高于正常结肠上皮细胞，而DUSP26蛋白表达及p-JNK/JNK、p-c-JUN/c-JUN水平均显著低于正常结肠上皮细胞（均P < 0.05）。CRC细胞来源的外泌体显著促进M2巨噬细胞极化，并上调下游蛋白p-Syk、p-PI3K、p-AKT、线粒体功能障碍蛋白DRP1和内质网应激蛋白CHOP、GRP78 蛋白表达（均P < 0.01）。DUSP26-OE 组或ST6GAL1-KO 组CRC 细胞来源的外泌体可抑制上述效应（P < 0.05或P < 0.01）。动物实验显示，ST6GAL1-KO组和DUSP26-OE组肿瘤体积均明显小于Vector组，联合干预组抑瘤效果最为显著（均P < 0.001）；联合干预组细胞增殖标志物PCNA 阳性率降低，TUNEL 阳性细胞显著增加（P < 0.05 或P < 0.001）。ST6GAL1-KO和DUSP26-OE可显著降低小鼠肿瘤组织GRP78和DRP1阳性比例及小鼠体内M2巨噬细胞比例。结论：CRC中ST6GAL1高表达可能通过影响外泌体唾液酸化水平，参与M2型巨噬细胞极化及应激相关蛋白表达调控，DUSP26低表达可能削弱JNK相关通路对ST6GAL1表达的负向调控；两者联合干预对移植瘤生长的抑制作用更明显。

Objective: To investigate the expression of β -galactoside α2, 6-sialyltransferase 1 (ST6GAL1) and dual-specificity phosphatase 26 (DUSP26) in colorectal cancer (CRC), and their effects on macrophage polarization and tumor progression. Methods: A total of 126 patients with CRC were enrolled. Immunohistochemical staining was used to detect the expression of ST6GAL1 and DUSP26 proteins in cancer tissues and adjacent tissues. Western blotting, co-immunoprecipitation, exosome characterization, flow cytometry, ELISA, and immunofluorescence were performed to analyze the effects of ST6GAL1, DUSP26, and related signaling pathways on CRC cell-derived exosomes, M2 macrophage polarization, mitochondrial function, and endoplasmic reticulum stress. ST6GAL1 knockout (ST6GAL1-KO) and DUSP26 overexpression (DUSP26-OE) cell models were established using CRISPR/Cas9 and lentiviral systems, respectively. A mouse tumor model was used to evaluate the effects of combined ST6GAL1 knockout and DUSP26 overexpression (ST6GAL1-KO + DUSP26-OE) intervention on tumor growth, cell proliferation, and apoptosis. Results: The positive rate of ST6GAL1 protein was significantly higher in CRC tissues than in adjacent normal tissues, whereas the positive rate of DUSP26 protein was significantly lower in CRC tissues than in adjacent normal tissues (both P < 0.001). ST6GAL1 protein expression was significantly higher in CRC cells than in normal colonic epithelial cells, whereas DUSP26 protein expression and the p-JNK/ JNK and p-c-JUN/c-JUN ratios were significantly lower than those in normal colonic epithelial cells (all P < 0.05). CRC cell- derived exosomes significantly promoted M2 macrophage polarization and upregulated the expression of the downstream proteins p-Syk, p-PI3K, and p-AKT, as well as the mitochondrial dysfunction-associated protein DRP1, and the endoplasmic reticulum stress- related proteins CHOP and GRP78 (all P < 0.01). Exosomes derived from DUSP26-OE or ST6GAL1-KO CRC cells inhibited these effects (P < 0.05 or P < 0.01). Animal experiments showed that tumor volume was significantly lower in the ST6GAL1-KO and DUSP26-OE groups than in the Vector group, and the combined ST6GAL1-KO and DUSP26-OE intervention group showed the most pronounced tumor-suppressive effect (all P < 0.001). In the combined intervention group, the positive rate of the cell proliferation marker PCNA was significantly decreased, whereas the number of TUNEL-positive cells was significantly increased (P < 0.05 or P < 0.001). ST6GAL1-KO and DUSP26-OE significantly reduced the positive rates of GRP78 and DRP1 and the proportion of M2 macrophages in mice. Conclusion: In CRC, high ST6GAL1 expression may participate in the regulation of M2 macrophage polarization and stress-related protein expression by affecting exosomal sialylation levels, while low DUSP26 expression may weaken the negative regulation of ST6GAL1 expression mediated by JNK-related pathways. Combined intervention targeting both factors exerted a more pronounced inhibitory effect on xenograft tumor growth.

黑龙江省卫生健康委科研课题（2020-397）