Stable suppression of beta-catenin expression in prostate cancer cell line by retrovirus mediated RNAi.
- Author:
Zheng HU
1
;
Fang CAI
;
Li-Juan CHENG
;
Kun XIA
;
Jia-Hui XIA
;
Zhuo-Hua ZHANG
Author Information
1. National Laboratory of Medical Genetics, Central South University, Changsha 410078, China.
- Publication Type:Journal Article
- MeSH:
Cell Line, Tumor;
Genetic Vectors;
Humans;
Male;
Prostatic Neoplasms;
metabolism;
pathology;
RNA Interference;
RNA, Small Interfering;
Retroviridae;
genetics;
Signal Transduction;
Wnt Proteins;
biosynthesis;
genetics;
beta Catenin;
biosynthesis;
genetics
- From:
Journal of Central South University(Medical Sciences)
2005;30(3):253-257
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis.
METHODS:We select 3 sites in the complete coden sequence region of beta-catenin gene as the RNAi targets, ligated the annealed double pre-DNA strands into the retroviral vectors pSUPER-retro and transfected them into the packaging cells PA317, and then collected supernatant with retrovirus to infect DU145. After selection by puromycin and culture expansion, the stable cell clones were attained. Expression of the 2 target genes of Wnt/beta-catenin signaling pathway cyclinD1 and c-myc, was detected in the beta-catenin RNAi cells by Western blot. The effect of suppressing beta-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium (MTT) assay.
RESULTS:Western blotting and RT-PCR showed that the expression level of beta-catenin in the 2 stable cell clones apparently decreased. CyclinD1 and c-myc expression decreased in the beta-catenin RNAi cells. MTT showed that the cell number of beta-catenin expression suppression cell clones decreased significantly (P < 0. 05), suggesting the cell proliferation was prevented.
CONCLUSION:The beta-catenin gene stable suppression cell line was successfully established.
