广西医学

目的分析对接蛋白3（DOK3）基因表达下调对原代结肠癌细胞增殖、侵袭/迁移能力的影响，并基于DOK3与G蛋白偶联受体84(GPR84）的相互作用探讨可能的作用机制。方法培养原代结肠癌细胞HCT16，将其分为对照组、小干扰RNA（siRNA）-NC组、siRNA-DOK3组进行实验。分别将siRNA-NC、siRNA-DOK3转染至siRNA-NC组、siRNA-DOK3组，而仅将培养液加入对照组。培养24 h后通过实时荧光定量PCR评估转染效果，再使用CCK-8法、Transwell实验分别检测各组细胞的增殖情况、侵袭/迁移能力，采用蛋白免疫共沉淀实验检测HCT16细胞中DOK3与GPR84之间的相互作用关系。结果与对照组比较，siRNA-DOK3组HCT16细胞中DOK3 mRNA表达量降低（P＜0.05），而siRNA-NC组HCT16细胞中DOK3 mRNA表达量差异无统计学意义（P＞0.05）。培养48 h、72 h后，siRNA-DOK3组HCT16细胞的增殖活力较对照组增加（P＜0.05）。与对照组相比，siRNA-DOK3组HCT16细胞的迁移和侵袭数量增加（P＜0.05），而siRNA-NC组HCT16细胞的迁移和侵袭数量差异无统计学意义（P＞0.05）。蛋白免疫共沉淀实验结果显示，在HCT16细胞中GPR84与DOK3相互结合。结论DOK3基因可能作为抑癌基因，参与了结肠癌的发生与发展，且其可能通过与GPR84结合发挥抑癌作用。

ObjectiveTo analyze the effect of down-regulated expression of docking protein 3 (DOK3) gene on proliferation, invasion/migration abilities of primary colon cancerous cells, and to explore its possible mechanism based on interaction effects between DOK3 and G protein-coupled receptor 84 (GPR84). MethodsPrimary colon cancerous cells HCT16 were cultured, and they were assigned to control group, small interfering RNA (siRNA)-NC group, or siRNA-DOK3 group, and then performing experiment. SiRNA-NC and siRNA-DOK3 were transfected into the siRNA-NC group and the siRNA-DOK3 group, respectively, whereas culture solution was only added into the control group. Transfection effect was evaluated by the real-time fluorescent quantitative PCR after 24 hours of culture, and then the CCK-8 method, Transwell experiment were used to detect proliferation, invasion/migration abilities of cells in various groups, respectively. Co-immunoprecipitation assay was used to detect interaction effects between DOK3 and GPR84 in HCT16 cells. ResultsCompared with the control group, the siRNA-DOK3 group exhibited a decreased mRNA expression of DOK3 in HCT16 cells (P＜0.05), whereas no statistically significant difference in DOK3 mRNA expression in HCT16 cells between the control group and the siRNA-NC group (P＞0.05). After 48- and 72-hour culture, proliferation activity of HCT16 cells in the siRNA-DOK3 group was increased as compared with the control group (P＜0.05). Compare with the control group, the siRNA-DOK3 group yielded increased number of HCT16 cell migrations and invasions (P＜0.05), whereas there was no statistically significant difference in the number of HCT16 cell migrations and invasions between the siRNA-NC group and the control group (P＞0.05). The results of co-immunoprecipitation assay revealed that GPR84 interacted with DOK3 in HCT16 cells.ConclusionDOK3 gene may be regarded as a tumor suppressor gene, which participates in the occurrence and development of colon cancer, and it may exert a tumor suppressive effect by binding with GPR84.

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