Influencce Of RNA Interference Targeting Ski Expression On Biologic Activity Of Osteosarcoma U-2OS Cells

Backgroud Osteosarcoma originated from mesenchymal tissue,joint system is the most common malignant tumor,the cure rate is low,the harm of adolescent health is higher,prone to lung metastasis in the early days,the mortality is higher.In recent years,with the development of the surgery and the wide application of neoadjuvant chemotherapy,the 5-year survival rate was significantly increased in patients with,but patients with metastatic 5-year survival rate is still low.Therefore,explore metastatic osteosarcoma mechanism,search for new therapeutic targets and extend is the lifetime of the shift problem which needs to be settled.Ski broad participation in different species and regulate a variety of cell proliferation and differentiation process,such as highly conserved in evolution process,participate in a variety of signaling pathways,and with the development of tumor,proliferation and differentiation.Existing research shows that Ski on a wide variety of tumor tissue is highly expressed in the state,the activation of Ski or abnormal expression is closely related to the development of tumor.And the Ski effect on osteosarcoma cell proliferation and migration of research at home and abroad,there is no related reports.Objective To investigate the effect of Ski specific siRNA on the biological activity of human osteosarcoma U-2OS cells in vitro.Then study the pathogenesis of osteosarcoma and provide theoretical basis for its early prevention,diagnosis and treatment.Methods The experiment was divided into three groups: blank control group(untransfected osteosarcoma cells),negative control group(transfection negative control sequence)and silent group(transfection Ski-siRNA)..The specific siRNA targeting Ski gene was transfected into U-2OS cells by Lipofectamine?RNAiMAX Reagent to establish Ski gene-silencing U-2OS cell line.Then the changes of Ski expression levels were detected by Western blotting.Cells’ proliferation vitality after Ski gene-silencing was measured by CCK-8 assay.Flow cytometry analysis of cellcycle;the migration ability after Ski gene-silencing was detected by wound healing assay.The changes of CyclinD1,matrix metalloproteinase-2/9 after Ski gene-silencing were detected by western blot.Results The Ski gene-silencing U-2OS cell line was successfully constructed.The expression level of Ski in U-2OS cells was significantly decreased after transfection with Ski-siRNA(P<0.05).The proliferation vitality of U-2OS cells at 12,24 and 48 hours after Ski gene-silencing were decreased than those in the blank control and negative control groups.Compared with the control group,the proportion of cell cycle G1 phase cells in the Ski-siRNA group was significantly increased,while the proportion of cells in the S phase and G2 phase was significantly reduced(p<0.05).In wound healing assay,the wound healing rate of Ski-siRNAgroup was significantly lower than that rate of the negative control siRNA group in 6、12、24、48 hours(P<0.05).Furthermore,the expression of PCNA,CyclinD1,MMP-2 and MMP-9were also suppressed in Ski-siRNA group(P<0.05).Conclusion Ski knocking down by siRNA significantly decreased the proliferation and migration ability of U-2OS cells.It is a reminding that Ski may become a new potential target therapy for human osteosarcoma disease.