Construction of a lentiviral vector of RNA interference against PPARS gene and the establishment of colon cancer cell line KM12C with stable knockdown of PPARdelta expression.
- Author:
Xiao JIANG
1
;
Lie YANG
;
Zongguang ZHOU
;
Yuan LI
;
Ling WANG
;
Yongyang YU
;
Cun WANG
Author Information
1. Institute of Digestive Surgery, West China Hospital, Sichuan Uniwversity, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Cell Line, Tumor;
Colonic Neoplasms;
genetics;
pathology;
Gene Knockdown Techniques;
Genetic Vectors;
genetics;
Humans;
Lentivirus;
genetics;
metabolism;
PPAR delta;
biosynthesis;
genetics;
RNA Interference;
RNA, Messenger;
biosynthesis;
genetics;
RNA, Small Interfering;
genetics
- From:
Journal of Biomedical Engineering
2010;27(2):400-406
- CountryChina
- Language:Chinese
-
Abstract:
Peroxisome proliferator-activated receptordelta (PPARdelta), as a downstream target of adenomatous polyposis coli (APC) signaling pathway, has been presumed to play some roles in colorectal carcinogenesis. However, the exact role of PPARdelta in colorectal cancer remains unclear. An HIV-1-based lentivirus packaging system was used for the construction of a lentiviral vector (lentivector) mediating RNA interference against PPARB. The direct sequencing demonstrated that the resulting lentivector containing the short-hairpin RNA expression cassette specifically targeting PPARdelta (sh-PPARdelta) was successfully constructed, and designated as pLVshPPARdelta. The control vector was designated as pLVControl. After the transduction, we observed highly efficient transduction (> 90%) of lentivirus in KM12C cells by fluorescent microscopy and fluorescence-activated cell sorting. Quantitative RT-PCR showed that pLVshPPARdelta lentivirus reduced PPARdelta mRNA expression by about 70.0% in KM12C cells as compared with that of the untreated cells (P < 0.05), while pLVControl had no significant effect on the PPARdelta mRNA level (P > 0.05). Western blot revealed an obvious reduction of PPARdelta protein expression in pLVshPPARdelta treated cells and showed no obvious difference between the control group and the untreated group. The results demonstrated that the lentivector mediating RNAi against PPARdelta was successfully constructed, which could stably knock down the PPARdelta expression in KM12C cells. This study finally provided a new cell model for the study of PPARdelta's function in colorectal cancer.
