Effect Of BMP9 Up-regulation And Id1 Down-regulation On Osteogenic Differentiation Of Osteosarcoma Cells And Related Mechanisms

PART ?:EFFECT OF INHIBITION OF ID1 GENE ON MALIGANT BEHAVIOR AND OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA CELLSObjective:To investigate the inhibition of Id1 gene on maligant behavior and osteogenic differentiation of osteosarcoma cells and its related mechanismsMethods:(1)The recombinant adenovirus Ad-si Id1 was used to treat osteosarcoma cell line MG63 to regulate Id1 expression,recombinant adenovirus Ad-RFP as a control group,MG63 without any virus treatment as a control group.(2)Semi-quantitative RT-PCR and western blot were used to determine the effect of adenovirus Ad-si Id1 and Ad-RFP on osteosarcoma MG63 cells at the expression of mRNA and protein respectively.(3)The apoptosis of osteosarcoma cells was detected by Annexin V-FITC single staining and DAPI staining,and the cell cycle distribution was detected by FCM.(4)The osteogenic differentiation of osteosarcoma was detected by ALP staining and calcium salt deposition.(5)The protein levels of Bcl-2 and Survivin,the key proteins of Wnt signaling pathway,?-catenin,ROR2 and CHOP were detected by western blot.Results:(1)The mRNA and protein expression of Id1 decreased(p<0.05)in MG63 cells that infected with recombinant adenovirus Ad-si Id1.(2)The apoptosis rate of osteosarcoma cells was significantly increased after silencing Id1 compared with the control group(p<0.05).(3)The ability of osteogenic differentiation of osteosarcoma was weak after down-regulating the expression of Id1.(4)The expression of anti-apoptotic factors Bcl-2 and Survivin were significantly down-regulated(p<0.05)after silencing Id1.(5)The expression of ?-catenin,ROR2 and CHOP of Wnt signaling pathway was down-regulated after silencing Id1(p<0.05).Conclusion:The recombinant adenovirus Ad-si Id1 can reduce the expression of Id1,and inhibition of Id1 expression can promote apoptosis of osteosarcoma cells and inhibit the expression of anti-apoptosis related genes Bcl-2 and Survivin.At the same time,Ad-si Id1 can promote early osteogenic differention,which may be related to the inhibition of wnt signaling pathway related gene expression.PART ?:EFFECT OF ID1 DOWN-REGULATION AND BMP9 UP-REGULATION ON OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA CELLS AND RELATED MECHANISMSObjective:To investigate the effect and mechanism of Id1 down-regulation combined with BMP9 up-regulation on osteogenic differentiation of osteosarcoma cells in vitro and in vivo;It provides a evidence for the treatment of osteosarcoma in the future.Metheds:(1)MG63 were induced with the recombinant adenovirus Ad-si Id1,Ad-BMP9,Ad-si Id1 combined with Ad-BMP9 and the control of recombinant adenovirus Ad RFP respectively.(2)Expression of Id1 and BMP9 protein in osteosarcoma cell MG63 were deteced by western blot.(3)In vitro,the early osteogenic differentiation were detected by alkaline phosphatase staining(ALP)and reading.The middle late stage osteogenic differentiation index(OPN)were detected by western blot,and calcium phosphate deposition test was used to detect the late osteogenic differentiation.(4)In vivo,tumor size and distant metastasis were observed by In Vivo Imagine Technology.H&E staining and Masson staining were used to observe the degree of osteogenic differentiation.(5)The expression level of Id1 and BMP9 in osteosarcoma cell line MG63 was regulated by recombinant adenovirus.After 24 h and 48 h,the expression of related signal pathway proteins was detected by western blot.Results:(1)Ad BMP9 can effectively increase the expression of BMP9(p<0.05),and the expression of BMP9 in BMP9+si Id1 group had no significant difference compared with BMP9+si Id1 group;the expression of Id1 in si Id1 group was reduced(p<0.05).The expression of Id1 in BMP9+si Id1 group was higher than that of other groups(p<0.05),and the expression of Id1 in BMP9 group had no significant difference campared with control group.(2)After over expression of BMP9 or inhibition of Id1 expression,ALP activity and ALP staining in osteosarcoma cells were higher than those in the control group(p<0.05),but the osteogenic differentiation ability of osteosarcoma cells was still weak.Inhibition of Id1 combined with BMP9 can effectively promote the alkaline phosphatase activity and staining of MG63(p<0.05).(3)The expression of OPN increased after infection of Ad-BMP9(higher than that of control group and si Id1 group).The expression of OPN was also higher than that of control group after inhibition of Id1,and the highest level of OPN expression was BMP9+si Id1 group.(4)The calcium salt deposition in group Adsi Id1 and Ad RFP group was not obvious,and there is a little calcium salt deposit in group Ad BMP9,but the most obvious calcium deposition was in the down-regulating Id1 combined with up-regulating BMP9 group.(5)In vivo,all groups were detected to be tumorigenic,and no distant metastasis was detected,there was no significant difference in tumor size;H&E staining and Masson staining showed that the osteogenic differentiation of the control group is not strong,alone inhibited Id1 or enhanced BMP9 expression can promote differentiation of osteosarcoma,but the osteogenic differentiation of BMP9+si Id1 group is the strongest.(6)The expression of p-AKT in group BMP9+si Id1 was not the strongest.The expression of p-AKT in group BMP9 was highest at 24 h and 48 h(p<0.01),while p-AKT expression in si Id1 group was not significantly different from that in blank control group at 24 h,but was higher than control group at 48 h.(7)For the expression of ?-catenin,BMP9 group and si Id1 group were lower than the BMP9+si Id1 group and the control group(p<0.01)at 24 h,BMP9 group was the highest at 48 h,while the strongest osteogenic differentiation group was lower than BMP9 group.(8)The expression of GSK-3? was the highest in group BMP9 at 24 h,and the lowest in the blank group.At 48 h,the expression of group si Id1 was not significantly different from that in the BMP9+si Id1 group,but the expression of the si Id1 group was lower at 24 h.Conclusion:(1)In vitro,ALP staining?ALP reading?the expression of OPN and calcium deposition were enhanced in down-regulating Id1 combined with down-regulating BMP9 group.However,the ability of osteogenic differentiation of osteosarcoma in early stage and late stage was weaker after overexpressing BMP9 alone.At the same time,the inhibition of Id1 expression alone can only promote the ALP staining and reading,indicating that the osteogenic differentiation of both of them was weaker than that of inhibition of Id1 combined with BMP9.(2)In vivo,there was no significant difference in the size of tumor,and no distant metastasis was found in the nude mice,but the osteoid matrix was increased in inhibition of Id1 combined with the BMP9 group.These results suggest that inhibition of Id1 combined with BMP9 can promote osteogenic differentiation of MG63.(3)By detecting the expression of the key factors in the related signaling pathways,the expression of related proteins in the BMP9+si Id1 group with the strongest osteogenic differentiation is in an intermediate state.It is indicated that osteogenic differentiation of osteosarcoma is a process of balancing through various signal pathways.The inhibition or increase of single signal pathway can not play a role in osteosarcoma.