Effects Of Combination Of DLX1 And BMP9 On Inducing Osteogeninc Differentiation Of Human Osteosarcoma Cells

PART Ⅰ The effect of DLX1 overexpression on malignant biological behavior and osteogenic differentiation of human osteosarcoma cellsObjective: To investigate the effect of DLX1 overexpression on malignant biological behavior and osteogenic differentiation of human osteosarcoma MG63 cells.Methods: Adenovirus adenocarcinoma(adenovirus,Ad)Ad DLX1 and Ad RFP were used to infect human osteosarcoma cells MG63,divided into DLX1 experimental group(Ad DLX1 infection group)and RFP control group(Ad RFP infection group).The expression of DLX1 was confirmed by RT-PCR and Western blot.Transwell assay was used to detect the migration and invasion of MG63 cells.Apoptosis of MG63 cells was detected by DAPI staining and flow cytometry.The proliferation of MG63 cells was detected by CCK-8.The expression of osteopontin(OPN)proteinwas detected by Western blot.Alizarin red staining was used to detect the late osteogenic ability of the cells.RT-PCR was used to study the related signal pathway,Western blot was used to verify the protein expression of the positive signal molecules.Results: 1.Ad DLX1 could significantly increased the expression of DLX1 in osteosarcoma MG63 cells compared with RFP control group after MG63 cells were infected by Ad DLX1 and Ad RFP,respectively.2.Compared with RFP control group,the cell migration ability and invasion ability of MG3 cells were decreased after DLX1 overexpression.3.After overexpression of DLX1 in MG63 cells,DLX1 had no significant effect on cell proliferation and apoptosis compared with RFP control group.4.After overexpression of DLX1 in MG63 cells,DLX1 had no significant effect on osteogenic differentiation of cells compared with RFP control group.5.Compared with RFP control group,DLX1 overexpression could up-regulate the expression of β-catenin in MG63 cells after overexpression of DLX1.Conclusions: 1.DLX1 overexpression have different effects on the malignant biological behavior of MG63 cells: DLX1 can inhibit the migration and invasion of MG63 cells,but have no significant effect on the proliferation and apoptosis of MG63 cells.DLX1 have no obvious effect on osteogenic differentiation of MG63 cells.2.After overexpression of DLX1 in MG63 cells,Wnt / β-catenin signaling pathway is involved in theregulation of DLX1 on the biological behavior of MG63 cells.PART Ⅱ Effects of DLX1 combined with BMP9 on malignant biological behavior and osteogenic differentiation of human osteosarcoma cellsObjective: To investigate the effect of DLX1 combined with BMP9 on malignant biological behavior and osteogenic differentiation of human osteosarcoma cell line MG63.Methods: Using recombinant adenovirus Ad DLX1 and AdBMP9,which construct DLX1 and BMP9 genes,alone or in combination Infected human osteosarcoma MG63 cells,the cells were divided into RFP group(Ad RFP group),BMP9 group(Ad BMP9 + Ad RFP infection group),DLX1 + BMP9 group(Ad DLX1 + Ad BMP9 infection group),DLX1 group(Ad DLX1 + Ad GFP infection group).The expression of DLX1 was confirmed by RT-PCR and Western blot.Transwell assay was used to detect the migration and invasion of MG63 cells.Apoptosis of MG63 cells was detected by DAPI staining and flow cytometry.The proliferation of MG63 cells was detected by CCK-8.The expression of osteopontin(OPN)protein was detected by Western blot.Alizarin red staining was used to detect thelate osteogenic ability of the cells.RT-PCR was used to study the related signal pathway,Western blot was used to verify the protein expression of the positive signal molecules.Results: 1.The expression of DLX1 m RNA and protein were up-regulated in MG63 cells after Ad DLX1 infection alone;the expression of BMP9 m RNA and protein were up-regulated in MG63 cells after Ad BMP9 infection alone;the expression of DLX1 and BMP9 were upregulated in MG63 cells after Ad DLX1 and Ad BMP9 were combined.2.Compared with RFP group,the migration and invasion of MG63 cells in BMP9 group,DLX1 + BMP9 group and DLX1 group were decreased,and the inhibition of migration and invasion in DLX1 + BMP9 group were higher than that in BMP9 group and DLX1 group.3.Compared with RFP group,the proliferative capacity of MG63 cells in BMP9 group and DLX1 + BMP9 group were decreased,and there was no significant difference in cell proliferation of DLX1 group.4.Compared with RFP group,there was no significant difference in apoptosis of MG63 cells between BMP9 group,DLX1 + BMP9 group and DLX1 group.5.Compared with RFP group,ALP activity,OPN protein expression and calcium salt deposition of MG63 cells in BMP9 group increased,but the degree of increase was limited.The activity of ALP 、the expression of OPN protein and he calcium salt deposition in MG63 cells were up-regulated significantly in DLX1 + BMP9 group.There was no significant increase in ALP activity,OPN proteinexpression and calcium salt deposition in DLX1 group.Conclusions: 1.DLX1 can inhibit the migration and invasion of MG63 cells,but has no significant effect on cell apoptosis and proliferation;DLX1 can not promote MG63 cell osteogenesis differentiation.2.BMP9 can inhibit the migration,invasion and proliferation of MG63 cells,and has no obvious effect on the apoptosis of MG63 cells.The promoting effect on osteogenesis differentiation of MG63 cells is limited.3.DLX1 combined with BMP9 can inhibit the migration,invasion and proliferation of MG63 cells,but have no significant effect on the apoptosis of MG63 cells.DLX1 combined with BMP9 can promote the osteogenic differentiation of MG63 cells.4.Osteosarcoma is a malignant proliferator caused by a differentiation disorder.DLX1 combined with BMP9 can reduce cell migration,invasion and proliferation,and promote osteogenic differentiation of human osteosarcoma cells.