Establishment of gastric cancer SGC-7901 cell line with stable lncRNA BC002811 silencing
10.16571/j.cnki.1008-8199.2019.04.006
- VernacularTitle: 稳定靶向干扰长链非编码RNA BC002811胃癌SGC-7901细胞株的构建
- Author:
Xiao-cong LIN
1
;
Xiao-yi CHEN
1
;
Hua-jun YU
1
;
Liu-bo LAN
1
;
Wei-yu FU
1
Author Information
1. Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang 524023, Guangdong, China
- Publication Type:Journal Article
- Keywords:
gastric cancer;
long non-coding RNA;
lentivirus;
short hairpin RNA
- From:
Journal of Medical Postgraduates
2019;32(4):364-368
- CountryChina
- Language:Chinese
-
Abstract:
Objective The aim of this study was to construct a recombinant lentivirus-mediated short hairpin RNA (shRNA) expression vector targeting the long non-coding RNA (lncRNA) BC002811 and establish a gastric cancer SGC-7901 cell line with stable BC002811 down-regulation. Methods Three small interfering RNAs (siRNA) were designed and synthesized. The best sequence for RNA interference was selected by real-time quantitative PCR (qPCR) and inserted into the lentiviral vector pLVX-shRNA2. After identification by DNA sequencing, the lentiviral vectors carrying BC002811 shRNA were packaged in HEK293T cells. The lentiviral particles were collected to infect human gastric cancer SGC-7901 cells. After screened by limiting dilution analysis, the SGC-7901 cell line with stable BC002811 down-regulation was established, the expression level of BC002811 detected by qPCR, and the effect of BC002811 on the proliferation of the cells analyzed by MTS. Results The results of qPCR showed that BC002811 siRNA-1 was the most effective siRNA sequence, with a knockdown efficiency of 87%. The recombinant lentiviral vector was packaged in the HEK293T cells with a viral titer of 3.7 × 108 TU/mL in the shRNA-1 group as compared with 4.5 × 108 TU/mL in the control. The expression of BC002811 in the shRNA-1 group was only 10% of that in the control group (P < 0.01), which indicated the successful establishment of the gastric cancer SGC-7901 cell line with stable BC002811 down-regulation. BC002811 knockdown significantly inhibited the proliferation of the SGC-7901 cells in the shRNA-1 group as compared with the control. Conclusion A recombinant lentiviral vector expressing BC002811 shRNA was successfully constructed and the gastric cancer cell line SGC-7901 with stable BC002811 silencing was established.
