Establishment of a hepatic carcinoma cell line with stable ClC-3 silencing by lentivirus-mediated RNA interference and its influence on invasion and migration
10.3969/j.issn.1001-1978.2015.08.015
- VernacularTitle: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
- Author:
Bin XU
;
Jialin LIN
;
Jingwen SHI
;
Shisi WANG
;
Jie YU
- Publication Type:Journal Article
- Keywords:
voltage-gated chloride channel 3;
hepatic carcinoma cells;
lentiviral vector;
RNA interference;
stable cell line;
migration;
invasion
- From:
Chinese Pharmacological Bulletin
2015;(8):1101-1107
- CountryChina
- Language:Chinese
-
Abstract:
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.
