Construction of RNA interference (RNAi) lentiviral expression vector of DEK gene and its effect on the biological behavior of liver cancer cells
10.3760/cma.j.issn.1007-3418.2020.03.000
- VernacularTitle:DEK基因RNA干扰慢病毒表达载体的构建及其对肝癌细胞生物学行为的影响
- Author:
Shuhua LI
1
;
Yu HOU
;
Zhe CHEN
;
Weiru WU
;
Chuanxin WU
;
Hang SUN
Author Information
1. 重庆医科大学病毒性肝炎研究所 重庆医科大学附属第二医院感染科 400010
- Keywords:
Proliferation;
Apoptosis;
Migration;
DEK;
RNA interference;
Lentiviral vector
- From:
Chinese Journal of Hepatology
2020;28(10):868-875
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct RNA interference (RNAi) lentiviral expression vector of DEK gene, and to explore its effect on the biological behavior of liver cancer cells.Methods:Double-stranded oligo DNAs were annealed and synthesized according to the interference sequence of DEK gene by RNAi technology. Small interfering RNA expression vector pLKO.1 was cloned after enzymatic digestion. The recombinant lentiviral pLKO.1-sh hDEK was constructed, and then the virus supernatant was collected, packed and infected by 293T cells. Real-time reverse transcription PCR (RT-PCR) and Western blot were used to detect DEK expression in human liver cancer cells Bel-7402, Hu-7, SmMC-7721 and HepG2, and DEK knockdown efficiency in each group of lentivirus-infected cells. Cell proliferation ability, cloning ability, apoptosis and migration ability were detected by cell counting kit-8 (CCK8), flow cytometry and scratch test, respectively. The t-test was used to compare the mean between the two groups, and one-way ANOVA was used to compare the multiple groups.Results:Enzymatic digestion and DNA sequencing results confirmed that the recombinant lentiviral vectors pLKO.1-sh hDEK1 and pLKO.1-sh hDEK3 were successfully constructed. RT-PCR and Western blot results showed that the expression of DEK in human liver cancer cells BEL-7402 and Huh7 cells was higher, and pLKO.1-sh hDEK3 was more effective in inhibiting the DEK gene expression ( P < 0.05). Therefore, pLKO.1-sh hDEK3 was selected to infect BEL-7402 and Huh7 cells for subsequent functional experiments. CCK8 cell proliferation test result showed that the cell proliferation ability of BEL-7402 and Huh7 cells infected with recombinant lentivirus was weakened when compared with blank control and negative control group ( P < 0.05). Apoptosis results showed that the apoptosis rate of knockdown group was higher than that of blank and the negative control group ( P < 0.05). Cell scratch test result showed that the wound healing rate of knockdown group was lower than that of blank control and negative control group ( P < 0.05), and the difference was statistically significant; however, there was no statistically significant difference between blank control and negative control group. Conclusion:Targeting DEK expression in silent liver cancer cells can inhibit the cell proliferation, migration ability, and induce apoptosis, which lays the foundation for further study of the role of DEK gene in liver cancer.
