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目的研究华支睾吸虫Severin蛋白对肝癌细胞株SMMC-7402增殖以及侵袭迁移能力的影响。方法构建Csseverin的慢病毒表达载体,并稳定转染肝癌SMMC-7402细胞。Western blot鉴定得到稳定过表达Csseverin的肝癌细胞系(PEZ-LV203-Csseverin 7402),并通过荧光显微镜观察其细胞内绿色荧光的表达;CCK-8实验检测过表达Csseverin后肝癌细胞SMMC-7402增殖能力的改变;用划痕愈合实验和Transwell小室侵袭实验检测Csseverin对SMMC-7402细胞迁移侵袭能力的影响;RT-PCR检测PEZ-LV203-Csseverin 7402和对照细胞内侵袭相关指标基质金属蛋白酶2(MMP-2)、MMP-9的m RNA水平。结果肝癌细胞SMMC-7402稳定过表达Csseverin后,Western blot检测出PEZ-LV203-Csseverin 7402中的Csseverin蛋白明显增多,荧光显微镜观察细胞有明显的绿色荧光表达;CCK-8实验表明Csseverin能明显促进SMMC-7402细胞的增殖能力。细胞划痕实验显示与对照细胞相比,过表达Csseverin的SMMC-7402细胞的迁移能力明显增强。Transwell小室检测显示,过表达Csseverin后的SMMC-7402中穿膜细胞数高于对照细胞组。RT-PCR检测出PEZ-LV203-Csseverin-7402中的MMP-2和MMP-9 m RNA水平明显高于对照细胞组。结论 Csseverin可促进肝癌细胞SMMC-7402的增殖,可能通过上调MMP-2和MMP-9表达从而促进肝癌细胞SMMC-7402的侵袭迁移能力。
Objective To study the effects of Clonorchis sinensis Severin protein on proliferation, invasion and migration of hepatocellular carcinoma cell line SMMC-7402. Methods The lentiviral vector of Csseverin was constructed and stably transfected into SMMC-7402 cells. Western blot was used to detect the expression of green fluorescence in cells stably expressing Csseverin (PEZ-LV203-Csseverin 7402). The CCK-8 assay was used to detect the proliferation of SMCC-7402 cells after Csseverin was overexpressed The effect of Csseverin on the invasion and migration of SMMC-7402 cells was detected by scratch healing test and Transwell chamber invasion assay. The expression of MMP-2 in PEZ-LV203-Csseverin 7402 and control cells were detected by RT- 2), m-RNA level of MMP-9. Results After Csseverin was stably overexpressed in SMMC-7402 hepatocellular carcinoma cells, Western blot showed that Csseverin protein in PEZ-LV203-Csseverin 7402 cells increased significantly, and green fluorescence was observed under fluorescence microscope. CCK-8 assay showed that Csseverin can significantly promote SMMC -7402 cells proliferation ability. Cell scratch assays showed that migration of Csseverin-overexpressing SMMC-7402 cells was significantly enhanced compared to control cells. Transwell chamber assay showed that the number of transmembrane cells in SMMC-7402 over-expressing Csseverin was higher than that in control cells. The level of MMP-2 and MMP-9 mRNA in PEZ-LV203-Csseverin-7402 was significantly higher than that in control cells by RT-PCR. Conclusion Csseverin can promote the proliferation of SMMC-7402 hepatoma cells, which may promote the invasion and migration of SMMC-7402 cells by up-regulating the expressions of MMP-2 and MMP-9.