SiRNA-mediated Notch2 Gene Silencing Inhibit The Invasion And Metastasis Of Human Osteosarcoma

Objective To investigate Notch2 expression in human osteosarcoma and discuss its role in human osteosarcoma.By silencing Notch2 gene targetly with siRNA,we try to explore its functions further and the mechanism.Method (1)We detected Notch2 expression by immunohistochemical methods in 82 cases of osteosarcoma tissues.And along with their clinical pathologic characteristics,we study the relationship between the Notch2 expression and their clinical pathologic characteristics.(2)We constructed full lenghth retrovirus pSUPER- Notch2-siRNA plasmid,and transfect it into the cultured PT67 cells to produce Notch2 siRNA and then silence Notch 2 expression in human osteosarcoma MG163 cells;In vitro,by wound healing assay,Transwell invasion test,MTT assay and flow cytometry analysis etc.methods,we discuss the molecular modulation mechanisms of Notch2 in the cell differentiation and proliferation and the invasion movement of human osteosarcoma to further verify the tumor biological behavior change regulated by Notch2 in human osteosarcoma.Result(1)Notch2 expression in human osteosarcoma Notch2 can be found in membrane and cytoplasm of the tumor cells,emerging as scat-tered brown stain distribution.In 82 selected cases,64 are positive,positive rate is 78.1%(64/82),while the control is 17.6%(6/34),and the difference was of statistical significance (P<0.05).This difference suggest that Notch2 participate in the develop-ment of human osteosarcoma.(2)The relationship betwwen the Notch2 expression and clinical pathologic characteristics.Notch2 expression is closely related to the differenciation, Ennecking staging and tumor metastasis of humon osteosarcoma (P<0.05).And have no relationship with the tumor size(diameter of the tumor),gender,age and ALP level(P>0.05).(3)Notch2 plays an important role in the invasion and metastasis potential of human osteosarcoma.Notch 2 mRNA and protein expression in MG63 cells.Contrast with control,Notch2 mRNA and protein expression level are 0.86±0.09 vs 0.47±0.05,0.76±0.04 vs 0.22±0.08 repectively,which have significant difference(P<0.05).(4) Notch2 expression in retrovirus siRNA specifically silenced MG63 cells Notch2 expression is obviously inhibited,inhibition efficiency is more than 70% compared to control(P<0.05),and Notch2RNA expression in control MG63 cells have no change,which suggest retrovirus mediated siRNA method can efficiently suppress the expression of Notch2 in MG63 osteosarcoma cells.(5)In vitro inhibition of Notch2 can influence the invasive capability of osteosarcoma cells.The migration of MG63Notch2RNAi+ cells is significantly decresed,24 hour wound healing rate are 52% vs 75%,with statistical difference(P<0.05).By Transwell Transwell invasion test,we found invasion ability of MG63Notch2RNAi+ cells is dramatically declined,and cell count of cells that tranverse the Transwell in low power field is 32.67±5.3 after 24 hours culture,which has significant difference compared with control(P<0.05).We detect the proliferation of MG63Notch2RNAi+ cells and the control by MTT method,and the results are (18.30±2.3)% vs (10±1.4)%,with statistical difference(P<0.05).Conclusion High expression of Notch2 in osteosarcoma may have something to do with the development of osteosarcoma, suggesting that it can be used as a tumor marker of poor prognosis in osteosarcoma; Notch2 along with its upstream and downstream signal molecular mechanism may participate in the invasion and metastasis of this tumor;Notch2 can be a new anti-osteosarcoma therapeutic target;Our reseach now provide an initial evidence of siRNA therapy of human osteosarcoma.