DOK3基因表达下调对原代结肠癌细胞生物学行为的影响及其可能机制  

Effect of down-regulated expression of DOK 3 gene on biologicalbehavior of primary colon cancerous cells and its possible mechanism

作　　者：孟云超[1] 陆正峰 胡凌佼 周静雾 邓秋芳 张启芳[1] MENG Yunchao;LU Zhengfeng;HU Lingjiao;ZHOU Jingwu;DENG Qiufang;ZHANG Qifang(Department of Gastroenterology,Nanxishan Hospital of Guangxi Zhuang Autonomous Region,Guilin 540021,Guangxi,China;Department of Gastroenterology,Minzu Hospital of Guangxi Zhuang Autonomous Region,Nanning 530001,Guangxi,China;Experimental Center for Medical Science,Nanxishan Hospital of Guangxi Zhuang Autonomous Region,Guilin 540021,Guangxi,China)

机构地区：[1]广西壮族自治区南溪山医院消化内科,广西桂林市540021 [2]广西壮族自治区民族医院消化内科,广西南宁市530001 [3]广西壮族自治区南溪山医院医学科学实验中心,广西桂林市540021

出　　处：《广西医学》2023年第23期2855-2860,共6页Guangxi Medical Journal

基　　金：广西医疗卫生适宜技术开发与推广应用项目(S2022026);广西壮族自治区卫生健康委员会自筹经费科研课题(Z20200526);广西医疗卫生重点培育学科建设项目(桂卫科教发[2023]1号)。

摘　　要：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DOK3mRNA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。Objective To 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).Methods Primary 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.Results Compared 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.Conclusion DOK3 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.

关 键 词：结肠癌 对接蛋白3 G蛋白偶联受体84 原代结肠癌细胞 恶性生物学行为 抑癌基因 

分 类 号：R735.35[医药卫生—肿瘤]