囊泡运输蛋白SNAP23对肿瘤细胞有丝分裂的调控作用

The effect of vesicle transport protein SNAP23 on the mitosis of tumor cells

目的研究囊泡运输蛋白SNAP23对肿瘤细胞有丝分裂的调控作用,为基于其细胞周期调控的抗肿瘤治疗策略提供理论基础。方法在HEK293T细胞中过表达细胞周期重要激酶CDK1的激活形式或灭活形式,检测二者是否会使SNAP23发生磷酸化修饰,并在肿瘤细胞中用两条不同序列的siRNA敲低SNAP23,采用Time lapse活细胞成像技术检测敲低SNAP23对有丝分裂期进程的影响,利用TCGA数据库分析SNAP23在肿瘤和癌旁组织的表达差异。结果 SNAP23能被激活形式的CDK1激酶复合体磷酸化,而不能被灭活形式的CDK1激酶复合体磷酸化;与对照敲低组比较,在肿瘤细胞中敲低SNAP23(SNAP23敲低组)可引起细胞有丝分裂的明显阻滞(P<0.01),其有丝分裂的前中期时间显著延长(P<0.01);TCGA数据库分析表明,与癌旁组织相比,SNAP23的mRNA表达量在胆管癌中明显升高(P<0.01)。结论囊泡运输蛋白SNAP23能被有丝分裂期重要激酶CDK1激酶复合体磷酸化,并调控肿瘤细胞的有丝分裂期进程。结合数据库分析,提示SNAP23的异常表达可能通过调控肿瘤细胞的细胞周期参与肿瘤的发生发展。

Objective To investigate the regulatory effect of vesicle transport protein SNAP23 on the mitosis of tumor cells,so as to provide a fundamental basis for the related anti-cancer therapy. Methods The HEK293 T cells were transfected with FlagSNAP23,kinase-active or inactive CDK1/Cyclin B1 complex,and the cell lysates were analyzed by immunoblotting to detect the phosphorylation of Flag-SNAP23. The HeLa cells were transfected with control siRNA or two different siRNAs targeting SNAP23,and then their mitotic progression was monitored by time-lapse imaging. The difference in the expression of SNAP23 between tumor tissues and tumor-adjacent tissues was analyzed based on the data in the TCGA database. Results SNAP23 could be phosphorylated by the kinase-active CDK1/Cyclin B1 complex,but not its kinase-inactive form. The depletion of SNAP23 induced obvious mitotic cycle arrest(SNAP23-depleted cells vs control cells,P<0.01),and the SNAP23 depletion also caused a prolonged prometaphase(SNAP23-depleted cells vs control cells,P<0.01). TCGA data analysis showed that the expression of SNAP23 was significantly higher in the cholangiocarcinoma tumor tissues than in the tumor-adjacent tissues(P<0.01). Conclusion Vesicle transport protein SNAP23 was phosphorylated by CDK1,and was required for the mitotic progression in tumor cells. Coupled with the result of TCGA data analysis,the present findings suggest that the abnormal expression of SNAP23 may participate in the tumorigenesis and tumor development via regulating the mitotic progression of cells.