Study On The Biological Effects And Mechanism Of SETD8 On Osteosarcoma Cell

Objective: To investigate the effects of siRNA reduction and SET domain(lysine methyltransferase)8(SETD8)gene expression on the proliferation,apoptosis,invasion and migration of osteosarcoma MG-63 cells and molecular mechanisms,and it is possible to regulate the above biological behavior of osteosarcoma MG-63 cells by inhibiting Wnt /β-catenin signal pathway.Methods:(1)To use the lipofection to transfect the siRNA of targeting the SETD8 gene into osteosarcoma MG-63 cells,this group is labeled as SETD8-siRNA group or experimental group.The heterologous siRNA is transfected into osteosarcoma MG-63 cells,this group is labeled as control-siRNA group or control group.For the MG-63 cells that are not transfected with siRNA,the group is labeled as un-transfected group or blank group.To use the CCK-8 cytotoxic activity assay to detect the proliferation of the above three groups of osteosarcoma MG-63 cells and use the plate cloning method to detect the cloning of MG-63 cells in each group,and use the AO-EB Fluorescence Double Staining Method to detect the apoptosis of each group.To use the real-time fluorescence quantitative PCR method to detect the mRNA expression levels of the proliferation and apoptosis of related moleculesβ-catenin,cyclinD1,caspase-9 and bcl-2 in MG-63 cells,and use the Western blotting method to detect the protein expression of the above molecules in MG-63 cells.(2)To use the lipofection to transfect the siRNA of targeting the SETD8 gene into osteosarcoma MG-63 cells,this group is labeled as SETD8-siRNA group or experimental group.The heterologous siRNA is transfected into osteosarcoma MG-63 cells,this group is labeled as control-siRNA group or control group.For the MG-63 cells that are not transfected with siRNA,the group is labeled as un-transfected group or blank group.To use the scratch healing method to detect the migration ability of the above groups of MG-63 cells,and use the transwell cabinet method to detect the invasion and migration of MG-63 cells in the three groups.To use the real-time fluorescence quantitative PCR method to detect the mRNA expression levels of theinvasion and migration of related molecules in MG-63 cells,and use the Western blotting method to detect the protein expression of the above molecules in MG-63 cells.Results:(1)After transfection of SETD8 gene into osteosarcoma MG-63 cells,the mRNA and protein expression of SETD8 gene in the experimental group are down-regulated comparing with the control group and the un-transfected group(P<0.05).The results of CCK-8 show that the proliferation of cells in the SETD8-siRNA group is significantly lower than that in the control-siRNA group and the un-transfected group(P<0.05).The results of plate cloning show that the self-cloning ability of SETD8-siRNA group is significantly lower than that in control-siRNA group and un-transfected group(P<0.05).The results of AO-EB staining show that the apoptosis of the SETD8-siRNA group is significantly higher than that in the control-siRNA group and the un-transfected group.In addition,the real-time fluorescence quantitative PCR experiments show that the mRNA expression levels of β-catenin,cyclinD1 and bcl-2 in the experimental group are significantly lower than those in the control group and the blank group(P<0.05),while the mRNA expression level of caspase-9 is significantly higher than that in the control group and the blank group(P<0.05).The results of Western blotting show that the relative expression levels of β-catenin,cyclinD1 and bcl-2 protein in SETD8-siRNA group are significantly lower than those in control-siRNA group and un-transfected group(P<0.05),while caspase-9 the expression level of protein is significantly increased(P<0.05).(2)Scratch healing experiments show that the migration ability of osteosarcoma cells in the SETD8-siRNA group is significantly lower than that in the other two groups(P<0.05).The results of transwell cabinet method show that the invasion and migration ability of MG-63 cells in the experimental group is significantly lower than that in the control group and the blank group(P<0.05).What’s more,the real-time fluorescence quantitative PCR experiments show that the relative mRNA expression levels of vimentin and mmp-3 in the SETD8-siRNA group are significantly lower than those in the control-siRNA group and the un-transfected group(P<0.05),the corresponding vimentin and mmp-3 protein in the experimental group are also much lower than those in the control group and the blank group(P<0.05).Conclusion:(1)Down-regulation of SETD8 gene expression can inhibit the proliferation ability of osteosarcoma MG-63 cells and can induce apoptosis inosteosarcoma MG-63 cells.(2)Down-regulation of SETD8 gene expression can inhibit the invasion and migration of osteosarcoma MG-63 cells.(3)The SETD8 gene may regulate the above biological behavior of osteosarcoma MG-63 cells through the Wnt/β-catenin signal pathway.