SND1低表达宫颈癌CasKi细胞稳定株的构建及其生物学功能变化

Construction of stable cervical cancer CasKi cell line with low expression of SND1 and its biological function changes

目的构建SND1低表达的人宫颈癌CasKi细胞稳定株,探讨SND1低表达对CasKi细胞生物学功能的影响。方法 CasKi细胞随机分为六组,空白对照组不做处理,阴性对照组、干扰1~4组分别转染TRC2-p LKO-puro Vector的空载慢病毒液及SND1 shRNA(1~4)慢病毒液,用嘌呤霉素筛选CasKi细胞稳定株。分别用Western blotting技术、RT-PCR技术检测CasKi细胞内SND1蛋白、mRNA表达,确认转染效率。用CCK-8法检测CasKi细胞增殖活力(OD_(450)值),用细胞划痕实验检测CasKi细胞相对迁移距离。结果空白对照组细胞全部死亡,阴性对照组及干扰1~4组细胞部分存活,证明细胞稳定株转染构建成功。干扰1~4组SND1蛋白、mRNA相对表达量与空白对照组、阴性对照组比较,P均<0.05;干扰3、4组与其他组比较,P均<0.05。干扰3、4组OD_(450)值、相对迁移距离与空白对照组、阴性对照组比较,P均<0.05。结论成功构建了SND1低表达的CasKi细胞稳定株;SND1低表达的CasKi细胞增殖和迁移能力降低。

Objective To construct a stable cervical cancer CasKi cell line with low expression of SND1 and detect the effect of SND1 on the biological function of cervical cancer cells. Methods CasKi cells were divided into six groups： the blank control group, negative control group, and interference groups 1-4. The cells in the negative control group and four interference groups were transfected with TRC2-pLKO-puro Vector empty lentivirus and SND1 shRNA（1-4）. The stable CasKi cells were screened out by puromycin. Western blotting and RT-PCR were used to detect the expression of SND1 protein and mRNA to confirm the transfection efficiency. The proliferation of CasKi cells was detected by CCK-8 （OD450）. The migration distance of CasKi cells was detected by cell Scratch test.Results Cells in the blank control group all die, and some in the negative control group and four interference groups survived, which indicated the stable cells were successfully constructed. Compared with the blank group and negative control group, the mRNA and protein expression levels of SND1 gene decreased in the four interference groups, and significant difference was found between the interference groups 3 and 4 （all P〈0.05）. Significant difference was found in the OD450 and migration between the blank group, negative control group, and the interference groups 3 and 4 （all P〈0.05）.Conclusion The stable CasKi cell line with low expression of SND1 is successfully established, and its proliferation and migration abilities decrease.