Inhibition Of Metastasis And Angiogenesis Of Osteosarcoma Cell Via Targeting Dll4 Gene In Vitro And Vivo By RNA Interference

Objective:To investigate the significance of Dll4 expressed in osteosarcoma tissues;construct targeted to silence the expression of Dll4 osteosarcoma invasion and metastasis and angiogenesis in rats.Methods:(1)The expression of Dll4 protein in 62 cases of osteosarcoma were detected by immunohistochemical method, combined with the clinical pathological data to analysis Dll4 expression relation to clinicopath olo gical characteristics.(2)By constructing full-length retroviral pSUPER-Dll4-siRNA plasmid,through the identification of post-its stable transfection PT67 cell culture to obtain virus serum protein secretion Dll4,intervention silence human osteosarcoma cell line SOSP-9607E10 Medium Dll4 expression, intervention silence the Department of Human microvascular endothelial cells(HMEC-1),in vitro experiments using scratches,Transwell invasion assay,blood vessels into a tube experiment,MTT and flow cytometry methods to study the regulation and control Dll4 osteosarcoma cell differentiation and proliferation,Osteosarcoma osteosarcoma invasion sports and vascular tube into the molecular mechanism of regulation and control to verify Dll4 osteosarcoma vascular tube into the molecular mechanisms leading to tumor biological behavior changes. Results:(1) The expression of Notch1 and Dll4 in osteosarcoma or osteochondroma.The positive expression of Notch1 protein in tumor cells mainly in the cytoplasm or cell membrane,showing brown, scattered.62 cases of osteosarcoma in 53 cases of positive expression, the positive rate was 85.5%,21.2%for the control group,the difference between the two was significant(P＜0.05).Dll4 protein expression mainly in the cytoplasm of tumor cells or cell membranes,some expression in vascular endothelial cells.62 cases of osteosarcoma in 55 cases of positive expression,the positive rate was 88.7%,23.6%for the control group,the difference between the two was significant(P＜0.05). Dll4 in osteosarcoma than in osteochondroma expression,suggesting that Notch1 and Dll4 may be involved in the occurrence of osteosarcoma, development.(2) The expression of Notch1 and Dll4 in osteosarcoma and its relationship with clinical indicators of the relationship between pathology.Notch1 and Dll4 expression in osteosarcoma exist a positive correlation between(P＜0.05),correlation coefficient rs for the 0.842,P =0.027.Notch1 and Dll4 protein expression and differentiation of osteosarcoma,Ennecking staging and metastasis are closely related(P＜0.05),but the diameter of tumor,gender,age,ALP(alkaline phosphatase) unrelated(P＞0.05). (3) The expression of Notch1 and Dll4 in osteosarcoma relationship to MVD.Notch1 high expression group MVD for 35.2±9.3, significantly higher than the low expression group 28.3±3.4,In addition,Dll4 high expression group MVD for 36.2±7.2,significantly higher than the low expression group 29.5±2.2,the difference was significant(P＜0.05).The results suggest that Notch1 and Dll4 protein expression and MVD between the closely related(P＜0.05),Notch1 and Dll4 protein expression in osteosarcoma may angiogenesis plays an important role in the process.(4) Dll4 at different metastatic potential osteosarcoma invasion of differentially expressed.Dll4mRNA and protein at SOSP-9607H9 and SOSP-9607E10 2 cells.The results showed that:At SOSP-9607H9 and SOSP-9607E 10 two osteosarcoma cell line cells,Dll4mRNA and protein expression level of significant difference,respectively,0.43±0.11 vs 0.86±0.09,0.47±0.05vs 0.82±0.08(P＜0.05).The results suggest: Dll4 expression in osteosarcoma is closely related to invasion and metastasis.(5) retrovirus-mediated small interference RNA silencing of specific cancer cell line SOSP-9607E10 Medium Dll4 expression.SOSP-9607E10^Dll4RNAi+ Medium Dll4 expression was significantly inhibited than the control group,its inhibition efficiencies in excess of 70%(P＜0.05),and as a contrast to SOSP-9607E10^Dll4RNAi-in Dll4 expression than the cell line SOSP- 9607E10-free change significantly.These results suggest that the present study using retroviral-mediated small RNA interference can effectively silence specific SOSP-9607E10 osteosarcoma cells in Dll4 expression.(6) in vitro inhibition of Dll4 expression can inhibit the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.SOSP-9607E10^Dll4RNAi+ and SOSP-9607E10^Dll4RNAi-cell migration capacity decreased 24-hour scratch healing rates were 62%vs 77%,difference statistically significant(P＜0.05).Using Transwell invasion chamber assay compared SOSP-9607E10^Dll4RNAi+ and SOSP-9607E10 ^Dll4RNAi- invasion capacity,results show that SOSP-9607E10^Dll4RNAi+ and SOSP-9607E10^Dll4RNAi-cell invasion decreased 24 hours after the train crossed the Transwell cell per low magnification field of vision respectively 63±11 vs 129±13,difference was significant(P＜0.05).Using MTT method SOSP-9607E10^Dll4RNAi+ and SOSP-9607E10 ^Dll4RNAi-proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant (P＞0.05).Thus the use of Annexin V and PI double staining method and flow cytometry detection of SOSP-9607E10^Dll4RNAi+ and SOSP-9607 E10 ^Dll4RNAi-in apoptosis,the results showed differences in two groups of cells in apoptosis is not significant(P＞0.05).These results suggest that in vitro inhibit the expression of Dll4 inhibits the invasion of osteosarcoma cell migration without affecting their proliferation and apoptosis.(7) in vivo inhibited the expression of Dll4 inhibits osteosarcoma tumor cells the ability.Comparison Mice^Dll4RNAi+ and Mice^Dll4RNAi-two nude mice model of SOSP-9607E10 primary tumor size,results showed that 45 days after subcutaneous plant SOSP-9607E10 primary tumor size (cm3) were 3.01±0.22 and 3.45±0.23,difference was significant(P＜0.05),compared MiceDll4RNAi + and Mice Dll4RNAi-two nude mice model of SOSP-9607E10 primary tumors of the MVD,showed Mice^Dll4RNAi+ and Mice^Dll4RNAi-two nude mice model of SOSP-9607E10 original hair tumors in each high-power microscope field of vision MVD values were an average of 29±2 and 37±3,difference significant(P＜0.05),which suggests the expression of Dll4 inhibition in vivo can inhibit the growth of osteosarcoma may be associated with inhibition of tumor angiogenesis relevant.(8) in vitro inhibition of Dll4 expression can inhibit HMEC-1 Human microvascular endothelial cell invasion without affecting the migration of its proliferation and apoptosis.Experimental group, HMEC-1^Dll4RNAi+ than the control group,HMEC-1^Dll4RNAi-cell migration capacity decreased 24-hour scratch healing rates were 69%vs 92%, difference statistically significant(P＜0.05).Cell invasion ability was significantly decreased 24 hours after culture through Transwell times each low vision cell count were 49±6 vs 70±3,difference significant (P＜0.05).MTT method HMEC-1^Dll4RNAi+ than the control group, HMEC-1^Dll4RNAi-proliferation ability,cell proliferation results showed discrepancies in the ability of the two groups is not significant(P＞0.05). Differences between two groups of apoptosis has no significant(P＞0.05).These results suggest that inhibition in vitro can inhibit the expression of Dll4 Human microvascular endothelial cells HMEC-1 invasion of migration without affecting their apoptosis.(9) in vitro in inhibiting the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis HMEC-1^Dll4RNAi+ endothelial cells in Matrigel formed should not mesh structure;control the composition of control index for 2397±726,the experimental index for the composition of tube 489±215,with differences between the two groups was significant(P＜0.05).Three-dimensional angiogenesis model control group and experimental group in each of 10 Microcarrier into possession of a few were 45±9 and 9±7,with difference significant(P＜0.05),These results indicate that in vitro inhibit the expression of Dll4 inhibits HMEC-1 Human microvascular endothelial cell angiogenesis.(10) in vivo in inhibiting the expression of Dll4 inhibits VEGF-induced angiogenesis.Experimental group in Matrigel neovascularization than the control group,experimental group and control group in Matrigel neovascularization separately for the total length of 23634±6421μm and 32145±4314μm,difference statistically significant(P＜0.05) This shows that in vivo inhibition Dll4 expression can inhibit VEGF-induced angiogenesis.Conclusion:Dll4 in osteosarcoma high expression may be involved in the occurrence and development of osteosarcoma,Dll4 may be used as prediction of poor prognosis in osteosarcoma tumor markers;Dll4 may control blood vessels into the molecular mechanisms involved in control of osteosarcoma invasion and metastasis;Dll4 likely to become anti-osteosarcoma invasion and metastasis and angiogenesis in a new target for gene therapy.