Establishment of a stable nasopharyngeal carcinoma cell line with lentivirus-mediated RNA interference for EIF4G1 gene silencing.
- Author:
Lu-xia TU
1
;
Wei-yi FANG
;
Zhen LIU
;
Xin LI
;
Ying HE
;
Si-ming XIE
;
Kai-tai YAO
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line, Tumor; Eukaryotic Initiation Factor-4G; biosynthesis; genetics; Genetic Vectors; genetics; Humans; Lentivirus; genetics; metabolism; Nasopharyngeal Neoplasms; genetics; metabolism; RNA Interference; RNA, Messenger; biosynthesis; genetics; RNA, Small Interfering; genetics; Transfection
- From: Journal of Southern Medical University 2009;29(5):844-851
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA).
METHODSThe EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F, 6-10B, C666-1, CNE1, CNE2, HNE1, HONE1, and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells with constant expression of the EIF4G1-siRNA, the efficiency of EIF4G1 mRNA expression interference was determined using QRT-PCR.
RESULTSThe 8 NPC cell lines showed differential expression of EIF4G1 mRNA, among which 5-8F cells had the highest EIF4G1 expression. The recombinant lentivirus plasmid pLenti6/BLOCK-iT-DEST/EIF4G1-shRNA was successfully constructed and verified by PCR and sequencing. The EIF4G1 mRNA level of 5-8F cells infected with shRNA-EIF4G1 lentivirus was significantly reduced as compared with the negative control and the blank control cells.
CONCLUSIONThe recombinant lentivirus vector pLenti6/BLOCK- iT-DEST/EIF4G1-shRNA we constructed results in marked downregulation of EIF4G1 mRNA expression and constant expression of EIF4G1-siRNA after infection of 5-8F cells.
