Establishment of a nasopharyngeal carcinoma cell line with stable nestin silencing by lentivirus-mediated RNA interference.
- Author:
Chuan LI
1
;
Ji MA
;
Yue ZHANG
;
Liang AN
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma; Cell Line, Tumor; Gene Silencing; Genetic Vectors; Humans; Intermediate Filament Proteins; genetics; Lentivirus; genetics; Nasopharyngeal Neoplasms; genetics; pathology; Nerve Tissue Proteins; genetics; Nestin; RNA Interference; RNA, Messenger; genetics; RNA, Small Interfering; genetics
- From: Journal of Southern Medical University 2011;31(4):604-609
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish a nasopharyngeal carcinoma (NPC) cell line with stable nestin gene silencing induced by short-hairpin RNA (shRNA) interference.
METHODSThe nestin mRNA levels in 8 NPC cell lines were detected by real-time PCR and immunofluorescence assay. The recombinant lentiviral shRNA expression plasmid targeting nestin was packaged into mature lentivirus by 293T cells, and the supernatant containing the virus was harvested, concentrated and titrated. The best target for RNA interference was selected by real-time PCR. 5-8F cells was then infected by the recombinant lentiviral vector, and the expression of nestin in the cells was detected by Western blotting and real-time PCR.
RESULTSThe 8 NPC cell lines showed different nestin expression levels, among which 5-8F cells had the highest nestin expression. The recombinant lentiviral vector was successfully constructed and verified by PCR and sequencing. Nestin mRNA and protein levels was significantly reduced in 5-8F cells infected with shRNA-nestin lentivirus as compared with the negative control and the blank control cells.
CONCLUSIONUsing the recombinant lentiviral vector constructed, we have successfully established a 5-8F NPC cell line with stable nestin gene silencing.
