[ShRNA-mediated gene silencing of beta-catenin inhibits growth of human colon cancer cell Colo205 in vitro].
OBJECTIVE
To observe the gene silencing and disruption of WNT pathway mediated by the specific shRNA targeted against beta-catenin and its effect on cell proliferation of the human colon cancer cell line Colo205.


METHODS
The shRNA plasmid vectors against beta-catenin were constructed and transfected into Colo205 cells with Lipofectamine 2000. The expression of beta-catenin was detected by RT-PCR and Western blot. Immunofluorescence staining was also performed to detect the beta-catenin protein expression in cells. The cell proliferation inhibition was determined by MTT assay and soft agar colony formation assay.


RESULTS
The shRNA vectors targeted against beta-catenin were successfully constructed and efficiently suppressed the expression of beta-catenin mRNA and protein(P<0.05). The expression inhibition rates were 47.89% and 45.26% at the mRNA and protein level respectively. Immunofluorescence microscopy also demonstrated the inhibition of beta-catenin protein induced by these specific shRNAs. The MTT assay indicated that the specific shRNA resulted in significant inhibition of cell growth on the culture plates in time-dependent manner. At 72 h post-transfection, the cell viability of CAT group was 48.5%, which was significantly different as compared with that of blank control group's 91.3%(P<0.05). In the soft agar, there were 9, 46, 43 cell colonies in the CAT, blank, and negative control groups respectively, which were significantly different(P<0.05).


CONCLUSIONS
The specific shRNAs targeted against beta-catenin has a gene silencing effect and blocks the WNT signaling pathway, which can inhibit the growth of Colo205 cells.
Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
Wen-Sheng
WS
Huang
Hui
H
Peng
Ming
M
Tan
Jin-Ping
JP
Ma
Jian-Ping
JP
Wang
Ping
P
Lan
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D034622:RNA Interference; D015398:Signal Transduction; D014162:Transfection; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics*
D051153:Wnt Proteins; D051176:beta Catenin