慢病毒介导过表达组织蛋白酶S基因对肝癌MHCC97H细胞增殖和迁移的作用

摘要

目的：建立慢病毒介导过表达组织蛋白酶S （cathepsin S，Cat S）基因的肝癌细胞株，观察Cat S基因对肝癌MHCC97H细胞增殖和迁移的影响。方法利用Age I和EcoR I双酶切获得目的基因片段，构建靶向过表达Cat S基因的慢病毒载体pLVX-EGFP-3FLAG-Puro，通过人胚肾上皮293T细胞进行慢病毒制备并感染肝癌MHCC97H细胞。使用嘌呤霉素加压筛选，建立稳定过表达Cat S基因的肝癌细胞株。通过RT-PCR、Western blot、MTT、Transwell和划痕实验研究稳定过表达Cat S基因对肝癌MHCC97H细胞增殖和迁移的影响。结果成功包装靶向过表达Cat S基因慢病毒载体并感染肝癌MHCC97H细胞。慢病毒感染后，与空载对照细胞Mock-MHCC97H相比，慢病毒Cat S-MHCC97H细胞Cat S mRNA、Cat S蛋白及MMP-2蛋白表达量明显升高（P<0.05），细胞增殖、迁移、侵袭能力亦明显增强（P<0.05）。结论慢病毒介导的Cat S基因过表达可能通过上调MMP-2蛋白表达促进肝癌MHCC97H细胞增殖、转移，Cat S基因可能是治疗肝癌的一个潜在靶点，这为阐明肝癌增殖和转移的分子生物学机制及提高肝癌基因治疗提供了新的实验依据。%Objective To construct a lentivirus expression vector to drive stable Cat S overexpression in the hepatocellular carcinoma (HCC)cell line MHCC97H,and to investigate the effects of overexpression on cell proliferation and migration. Methods The Cat S gene was inserted into the lentiviral expression vector pLVX-EGFP-3FLAG-Puro using Age I and EcoR I restriction enzymes and transfected into 293T cells to generate recombinant lentivirus. This virus was used to infect MHCC97H cells,and positive cells were selected using puromycin. Real-time quantitative PCR,Western blots,MTT assay,the transwell migration assay and the wound healing migration assay were used to assess the effects of Cat S overexpression on proliferation and migration of MHCC97H cells. Results A lentiviral vector containing the Cat S gene was constructed,and an MHCC97H cell line stably overexpressing Cat S was established. Cat S overexpression was associated with significantly shorter cell doubling time,as well as significantly greater invasion and migration abilities. Conclusions Lentivirus-mediated Cat S overexpression can increase MHCC97H proliferationand migration,opening the door to future studies of molecular mechanisms of liver cancer invasion and metastasis.