OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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).

CONCLUSIONSThe 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.