The Effects Of Silencing SCO2 Gene On Proliferation And Apoptosis Of Breast Cancer Cells MCF-7 And Explore Its Underling Mechanism

Objective: To investigate the expression of synthesis of cytochrome c oxidase 2(SCO2)in breast cancer tissue and breast cancer cells,and to study the effect of silencing SCO2 gene on proliferation and apoptosis of MCF-7 cells by lentivirus vector in order to explore the underling mechanism.Methods:1.Immunohistochemical method was applied to detect the expression of SCO2 in breast cancer and adjacent paracancerous tissues.2.The silence effect on SCO2 gene mRNA levels and protein levels were detected by real-time PCR and Western Blot respectively in human normal mammary epithelial cell line MCF-10 A,breast cancer cells MCF-7 and MDA-MB-231.3.SCO2 gene coding region was cloned into lentivirus vector.Lentivirus particles were infected into MCF-7 cells to knock down the expression of SCO2 gene.The SCO2 gene silencing effect was verified by real-time fluorescence quantitative PCR and Western blot.4.CCK-8 assay and plate colony forming assay were used to detect the effects of silencing SCO2 on the proliferation of MCF-7 cells.Flow cytometry was used to detect The effects of silencing SCO2 on the cell cycle and apoptosis of MCF-7 cells.5.In order to explore the underling mechanism of silencing SCO2 on proliferation and apoptosis of MCF-7 cells,Western blot was used to detect the expression levels of Bax,Bcl-2,pro-caspase3 and cleaved-caspase3.Results:1.Immunohistochemistry showed that SCO2 gene was mainly expressed in the cytoplasm of breast cancer cells.SCO2 gene was low-expression in breast cancer tissues(P<0.05).2.qRT-PCR and Western blot assay showed that the relative expression of SCO2 mRNA and SCO2 protein were lower in breast cancer cells MCF-7 and MDA-MB-231 than what in human normal breast epithelial cells MCF-10 A,and its expression levels in MCF-7 cells were higher than that in MDA-MB-231 cells(P<0.05).3.The silencing effect was detected by real-time fluorescence quantitative PCR and Western blot respectively.The SCO2 expression in human breast cancer MCF-7 cells transfected with SCO2 lentiviral vector was significantly decreased(P<0.05).4.CCK8 showed that the proliferation ability of LV-shRNA-SCO2 cells was significantly higher than that of LV-shRNA-NC cells and MCF-7 cells(P<0.05).Plate clone formation assay showed that the cell clone formation of LV-shRNA-SCO2 cells was(53.20±3.88)%,which was higer than(41.67±4.25)% in LV-shRNA-NC cells and(40.50±1.32)% in MCF-7 cells(P<0.05).5.Flow cytometry results showed that cell percentage of G0/G1 stage in LV-shRNA-SCO2 cells was(67.48±0.69)%,which were lower than MCF-7 cells(71.20±0.72)% and LV-shRNA-NC cells(72.99±0.40)%,However,the percentage of cells in LV-shRNA-SCO2 cells at S phase was(28.01±0.93)%,higher than that in MCF-7 cells(22.27±1.71)% and LV-shRNA-NC cells(21.64±0.67)%(P<0.05).6.The results of flow cytometry showed that the apoptosis rate of LV-shRNA-SCO2 cells was(2.60 ± 0.40)% compared with that in MCF-7 cells(6.97±0.41)% and LV-shRNA-NC cells(6.87±0.11)% was lower(P <0.05).7.Study on related mechanism: Western blot results showed that the protein relative expression of Bax and Cleaved-caspase3 in LV-shRNA-SCO2 cells were lower than that in MCF-7 cells and LV-shRNA-NC cells,while he protein relative expression of Bcl-2 was higher than that in MCF-7 cells and LV-shRNA-NC cells(P<0.05).Conclusions: 1,The expressiom of SCO2 gene was lowly in breast cancer tissues and human breast cancer cells.2.Silencing SCO2 gene can enhance the proliferation of human breast cancer line MCF-7 cells,activate the cell cycle,promote the proliferation of breast cancer cells and inhibit its apoptosis.3.The effect of SCO2 gene on the proliferation and apoptosis of human breast cancer MCF-7 cells may be related to the apoptosis-related proteins Bcl-2,Bax and cleaved-caspase3.