MAD2基因沉默促进人食管鳞癌细胞系KYSE30细胞增殖和侵袭能力的实验研究

摘要

Background and purpose: Mitosis checkpoint defect could lead to chromosomal instability and distort biological behaviour of cells. This study was to explore the effect of silencing mitotic arrest deficient 2 (MAD2) with siRNA on the proliferation and invasion properties of esophageal squamous cell carcinoma cell line KYSE30. Methods: The eukaryotic expression vector of siRNA specific for MAD2 gene was constructed by liposome transfection, and the silencing effect was identified by RT-PCR and Western blot. According to the inhibitory effect of the siRNA vectors, the esophageal squamous cell carcinoma cells were divided into control group, non-specific MAD2 siRNA group and specific MAD2 siRNA group. The cell proliferation, migration and adhesion were finally detected in control group and MAD2 siRNA groups after 48 h. MTT and cloning forming assays were applied to detect the cell proliferation of all groups. Wound-healing and invasion assays were used to examine the abilities of migration and invasion, respectively. Results: For 4g-hour specific MAD2 siRNA group, the expression of MAD2 in the cells was the lowest. Accordingly, the cell viability was increased; the ability of migration and adhesion were also increased.Conclusion: Inhibition of MAD2 gene can enhance the abilities of proliferation and invasion in esophageal squamous cell carcinoma. These data have provided important insights into the probable functions of MAD2 protein in the process of tumor generation and metastasis.%背景与目的:有丝分裂检测点缺陷可引起细胞染色体不稳定性而使细胞生物学行为改变,本实验探讨RNA干扰有丝分裂阻滞缺陷蛋白2(mitotic arrest deficient 2,MAD2)基因表达对人食管鳞癌细胞系KYSE30细胞增殖和侵袭能力的影响.方法:利用脂质体转染的方法,将MAD2基因的siRNA转染食管鳞癌细胞KYSE30,并通过RT-PCR以及免疫印迹的方法进行siRNA效果鉴定.实验分为正常对照组、非特异干扰组、特异干扰组.MTT、克隆形成实验检测细胞增殖活性的变化,损伤刮擦实验和Transwell小室实验分别检测转染细胞株的迁移与侵袭能力.结果:siRNA作用48 h组,MAD2蛋白的表达最低;相应地,迁移黏附能力增强,侵袭实验显示转染后细胞侵袭能力较转染前有显著加强.结论:MA4D2基因沉默能够促进人食管鳞癌细胞系KYSE30的体外增殖和侵袭能力增强,并抑制凋亡,提示其变化对肿瘤的发生和转移发挥重要作用.