慢病毒介导的干扰ClC-3肝癌稳定细胞株的建立及其侵袭和迁移能力的改变

摘要

目的建立慢病毒介导的靶向干扰电压门控氯通道3(voltage-gated chloride channel 3,ClC-3)的稳定肝癌细胞株,并探讨其侵袭和迁移能力的改变。方法构建靶向 ClC-3的3个短发夹状RNA ( short-hairpin RNA, shRNA)慢病毒表达载体,293FT细胞包装慢病毒并测定滴度。重组慢病毒感染肝癌细胞株MHCC97H,筛选稳定干扰细胞株。实时荧光定量PCR和Western blot检测ClC-3 mRNA和蛋白的表达以确定干扰效率。 Transwell小室实验(含Matrigel和不含Ma-trigel)和细胞划痕实验检测ClC-3基因被抑制后侵袭和迁移能力的改变。结果成功获得4种慢病毒,感染MHCC97细胞后,建立1株阴性对照细胞和3株 ClC-3稳定干扰细胞( MHCC97H/shClC-3-1、shClC-3-2和 shClC-3-3)。3株稳定干扰细胞ClC-3 mRNA和蛋白表达水平明显低于阴性对照细胞(P<0．01),MHCC97H/shClC-3-2细胞对ClC-3的抑制效果最好。与阴性对照细胞相比,MHCC97H/shClC-3-2细胞侵袭和迁移能力都下降( P<0．01)。结论成功建立稳定干扰ClC-3基因的肝癌细胞株,ClC-3表达抑制会降低MH-CC97H细胞的侵袭和迁移能力。%Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.