| ObjectiveTo study the effect of the Notch signaling pathway for the biological behaviors such as proliferation,apoptosis,migration,invasion ability of MG63 and the early and late-stage osteogenic differentiational ability of MG63.Methods1.RT-PCR was used to verify the expressional differences of the Notch signaling pathway related genes between normal human osteoblasts and different human osteosarcoma cells;2.The classic inhibitor of the Notch signaling pathway DAPT was used to inhibit the Notch signaling pathway of MG63(DAPT,inhibiting the Notch signaling pathway group),the recombinant adenovirus of overexpressing Jagged1 was used to activate the Notch signaling pathway of MG63(Ad-Jagged1,activating the Notch signaling pathway group),the empty recombinant adenovirus was used as the adenovirus Control group(Ad-RFP group),DMSO was used as the co-solvent control group(DMSO group),and MG63 without any treatment was used as the blank control group(Control group);3.RT-PCR was used to verify the gene level of the Notch signaling pathway after were inhibited by DAPT(?-secretase inhibitor)and activated by Ad-Jagged1(overexpress Jagged1 adenovirus)of MG63;4.CCK8 was used to detect the proliferation ability of MG63 after regulating the Notch signaling pathway;5.DAPI staining was used to detect the apoptosis level of MG63 after regulating the Notch signaling pathway;6.Scratch and Transwell were used to detect the migration and invasion ability of MG63 after regulating the Notch signaling pathway;7.Alkaline Phosphatase and Alizarin Red Staining were used to detect the early and late-stage osteogenic differentiation ability of MG63 after regulating the Notch signaling pathway;8.RT-PCR was used to verify the expressional level of differentiation inhibitor 1(Id1)of MG63 after regulating the Notch signaling pathway.Results1.RT-PCR results showed that key genes of the Notch signaling pathway DLL1,Notch1,Jagged1,Hes1 and HEY1 were abnormally expressed in osteosarcoma cells(P<0.05);2.Inhibited the Notch signaling pathway was inhibited by DAPT(P<0.05),and the Notch signaling pathway was activated by Ad-Jagged1(P<0.05);3.Inhibition of the Notch signaling pathway could inhibit the ability of proliferation,migration and invasion of MG63,and induce its apoptosis(P<0.05);Activation of the Notch signaling pathway could promote the ability of proliferation,migration and invasion of MG63,and inhibit its apoptosis(P<0.05);4.Inhibition of the Notch signaling pathway could inhibit the osteoblast differentiation of MG63(P<0.05);Activation of the Notch signaling pathway could promote the osteogenic differentiation of MG63(P<0.05);5.The expression of differentiation inhibitor 1(ID1)in MG63 was decreased after the Notch signaling pathway was inhibited(P<0.05);the expression of differentiation inhibitor1(ID1)in MG63 was increased after the Notch signaling pathway was activated(P<0.05).Conclusion1.The Notch signaling pathway genes level were abnormally high of human osteosarcoma cells;2.Inhibition of the Notch signaling pathway could reverse the malignant biological behavior and inhibit the osteogenic differentiation of MG63;Activation of the Notch signaling pathway could enhance the malignant biological behavior and promote the osteogenic differentiation of MG63;3.The Notch signaling pathway could influence the expressional level of Id1 of MG63.Objective To study the effect of the Notch signaling pathway combined with Id1 on the malignant biological behaviors such as proliferation?apoptosis?migration and invasion of MG63 and the early and late-stage osteogenic differentiational ability of MG63.Methods 1.Western blot was used to detect the Id1 protein expression level of MG63 in the following groups(MG63 without any treatment as blank control,MG63 was treated by DAPT as the group of inhibiting the Notch signaling pathway,MG63 was treated by Ad-Jagged1 as the group of activating the Notch signaling pathway,MG63 was treated by Ad-si Id1 as the group of silencing Id1,MG63 was treated by Ad-Id1 as the group of overexpressing Id1,MG63 was treated by DAPT+Ad-si Id1 as the group of inhibiting the Notch signaling pathway combined with silencing Id1,MG63 was treated by DAPT+Ad-Id1 as the group of inhibiting the Notch signaling pathway combined with overexpressing Id1,MG63 was treated by Ad-Jagged1+Ad-si Id1 as the group of activating the Notch signaling pathway combined with silencing Id1,MG63 was treated by Ad-Jagged1+Ad-Id1 as the group of activating the Notch signaling pathway combined with overexpressing Id1);2.CCK8 and Crystal Violet Stain were used to detect the proliferation ability of MG63 after regulating the Notch signaling pathway combined with Id1;3.DAPI staining was used to detect the apoptosis level of MG63 after regulating the Notch signaling pathway combined with Id1;4.Scratch and Transwell were used to detect the migration and invasion ability of MG63 after regulating the Notch signaling pathway combined with Id1;5.Alkaline Phosphatase and Alizarin Red Staining were used to detect the early and late-stage osteogenic differentiation ability of MG63 after regulating the Notch signaling pathway combined with Id1.Results 1.Compared with the control group,the protein level of Id1 in DAPT and Ad-si Id1 group were down-regulated,the protein level of Id1 in Ad-Jagged1 and Ad-Id1 group were up-regulated(P<0.05);compared with the DAPT group,the protein level of Id1 in DAPT+Ad-si Id1 group was down-regulated,the protein level of Id1 in DAPT+Ad-Id1 group was up-regulated(P<0.05);compared with the Ad-Jagged1 group,the protein level of Id1 in Ad-Jagged1+Ad-si Id1 group was down-regulated,the protein level of Id1 in Ad-Jagged1+Ad-Id1 group was up-regulated(P<0.05);2.Inhibition the Notch signaling pathway or silencing Id1 could inhibit the proliferation?migration and invasion ability of MG63 but could induce its apoptosis level;compared with the inhibiting Notch signaling pathway group,inhibiting the Notch signaling pathway combined with silencing Id1 could inhibit the proliferation?migration and invasion ability of MG63 but could induce its apoptosis level further,inhibiting the Notch pathway combined with overexpressing Id1 could promote the proliferation?migration and invasion ability of MG63 but could reduce its apoptosis level(P<0.05);Activating the Notch signaling pathway or overexpressing Id1 could promote the proliferation?migration and invasion ability of MG63,but reduce its apoptosis level;compared with the activation of the Notch signaling pathway group,activating the Notch pathway combined with silencing Id1 could inhibit the proliferation?migration and invasion ability of MG63 but could induce its apoptosis level,activating the Notch signaling pathway combined with overexpressing Id1 could promote the proliferation?migration and invasion ability of MG63 but could reduce its apoptosis level further(P<0.05);3.Inhibition of the Notch signaling pathway could inhibit the osteogenic differentiation,but silence Id1 and inhibiting the Notch signaling pathway combined with silencing Id1 could promote the osteogenic differentiation of MG63,in which,the ability of osteogenic differentiation in DAPT+Ad-si Id1 group was the strongest(P<0.05);compared with activation of the Notch signaling pathway could promote the osteogenic differentiation,activating the Notch pathway combined with silencing Id1 could promote the osteogenic differentiation of MG63 and activating the Notch signaling pathway combined with overexpressing Id1 could inhibit the osteogenic differentiation of MG63(P<0.05).Conclusion 1.The Notch signaling pathway combined with Id1 could influence the malignant biological behavior and osteogenic differentiation of MG63;2.Inhibiting the Notch signaling pathway combined with silencing Id1 could weaken the malignant biological behavior and promote the osteogenic differentiation. |
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