Effects Of RNA Interference-mediated β-catenin Silencing On MG63 Osteosarcoma Cell Line

PartⅠEffect of decreasedβ-catenin expression by siRNA on the proliferation and apoptosis of osteosarcoma cellObjectiveβ-catenin,the chef oncogenic component of the canonical Wnt pathway, is known to be involved in a variety of cancer. However, the role ofβ-catenin in human osteosarcoma has not been fully understood. Here we investigate the effect of small intering RNA-mediated P-catenin knockdown on the survival and apoptosis of human osteosarcoma cell line MG-63.Methods The siRNA againstβ-catenin was constructed and transfected into MG-63 cells with Lipofectamine TM2000. The expression ofβ-catenin was detected by Realtime-PCR and Western blot. Flow cytometry was used to detect the cell apoptosis by Annexin V-FITC/PI staining. The proliferation of cells was assayed by CCK-8 method.Results Compared with the control group, the mRNA and protein level was significantly decreased in experimental group transfected withβ-catenin SiRNA. Cell apoptosis analysis revealed no obvious change in MG-63 cell line. CCK-8 assay revealed no change.Conclusion siRNA against P-catenin is effective, but there is no significant change on apoptosis and proliferation of MG63 osteosarcoma cell line. Part II SiRNA-mediated silencing ofβ-catenin suppresses invasion in MG-63 osteosarcoma cellsObjective To investigate the effect of down-regulation ofβ-catenin gene expression on cell invasion and motility in osteosarcoma cell line MG63.Methods The siRNA againstβ-catenin was transfected into MG-63 cells with Lipofectamine TM2000. Transwell test was used to detect the invasion ability of MG-63 cells. The expression of MTl-MMP was detected by Western blot.Results We observed a significant decrease in invading capacity ofβ-catenin siRNA-transfected cells. The number ofβ-catenin siRNA transfected cells (25.45±4.28, p <0.05) passing through the Matrigel was markedly lower than the numbers of negative control (101.57±15.03) and control (110.16±14.35) cells. p-catenin siRNA transfected cells were significantly less motile than negetive control and control cells. compared with control groups, the expression of MTl-MMP were found to be reduced significantly.Conclusion Knock-down ofβ-catenin gene may decrease the invasion ability through down-regulated MTl-MMP expression Part III SiRNA-mediated silencing ofβ-catenin suppresses chemosensitivity to doxorubicin in MG-63 osteosarcoma cellsObjective To explore the effect of down-regulation ofβ-catenin gene expression on cell chemosensitivity to doxorubicin in osteosarcoma cell line MG63.Method Detection of apoptosis by flow cytometry was performed using the Annexin V-FITC-PI kit. Chemosensitivity to Doxorubicin of MG-63 cells were determined by CCK-8. Western-blot was used to analysis the level of p65, XIAP and Bclxl expression. Realtime PCR was performed to detect the NF-kappaB target gene XIAP and Bclxl mRNA expression.Results With flow cytometry, when exposed to doxorubicin,β-catenin-siRNA transfected cells were much more resistant to apoptosis than negetive control and control cells. The cellular apoptosis rate was 13.06±1.34%*,27.74±2.52%and 29.64±1.63% inβ-catenin-siRNA group, negative control group and control group, respectively,*P<0.05. Using CCK-8 assay, the growth of cells was measured after 24h of exposure to a range of concentrations of doxorubicin, cells transfected withβ-catenin siRNA were less sensitive to doxorubicin than controls. the mRNA level of Bclxl and XIAP were significantly inreased by 2.1-and 1.8-fold compared to control, respectively. And western blot analysis also demonstrated significant increase inβ-catenin siRNA treated cells compared to control cells.Conclusion siRNA mediated downregulation ofβ-catenin enhance XIAP, Bclxl and nuclear p65 expression, thereby resulting in reduction of chemosensitivity to doxorubicin in vitro.