Effects of cyclophilin D-mediated mitochondrial permeability transition pore opening on the biological behavior of osteosarcoma cells
Aim:To investigate the effects of interference with the key mitochondrial protein cyclophilin D(CypD)on the proliferation,migration and apoptosis of osteosarcoma MG63 cells.Methods:Three MG63 cell lines transfected with dif-ferent siRNA knockdowns of CypD were constructed,namely CypD-siRNA1,CypD-siRNA2 and CypD-siRNA3 groups,and the expression of CypD in the 3 groups was detected by qRT-PCR and Western blot,and CypD-siRNA was screened for sub-sequent experiments.MG63 cells were divided into negative control group(transfected with siRNA-NC)and knockdown group(transfected with CypD-siRNA2).The proliferation ability of MG63 cells was detected by CCK-8 assay and plate clo-ning assay,and the migration ability of MG63 cells was detected by Transwell chamber assay.MitoSOX Red was used to de-tect the content of mitochondrial reactive oxygen species,and calcein and cobalt were used to evaluate the open state of mi-tochondrial permeability transition pores.The apoptosis was detected by flow cytometry.The expressions of proteins related to migration and apoptosis of MG63 cells were detected by Western blot.Results:Compared with CypD-siRNA1 and CypD-siRNA3 group,the mRNA and protein expression levels of CypD in CypD-siRNA2 group were decreased(P<0.05).Com-pared with negative control group,the number of clones and migration cells and proliferation ability decreased,apoptosis rate increased,N-cadherin and Vimentin protein expression levels decreased(P<0.05),and Bax/Bcl-2,Cleaved Caspase 3/Caspase 3,E-cadherin and Cytochrome C protein expression levels increased(P<0.05).The level of mitochondrial reactive oxygen species in knockdown group was higher than that in negative control group,and the openness of mitochondrial perme-ability transition pore was lower than that in negative control group(P<0.05).Conclusion:CypD knockdown can up-regu-late the oxidative stress level,inhibit proliferation and migration ability,and induce apoptosis of MG63 cells through mito-chondrial pathway.
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维普
