SPAG6 Mediates Cytosolic Localization And Phosphorylation Of P27^kip1 To Regulate Proliferation And Apoptosis Of Ovarian Cancer Cells

Objective:To explore the effect of sperm-associated antigen 6（SPAG6）on the proliferation and apoptosis of ovarian cancer cells and its regulatory mechanism.Methods:The expression of SPAG6 gene in normal human ovarian epithelial cells NOE-71 and human ovarian cancer cells OVCAR-3 and SKOV-3 cells was detected by real-time fluorescent quantitative PCR.Four"SPAG6-sh RNA"plasmids which potentially inhibit the expression of SPAG6 were transfected into ovarian cancer cells SKOV-3,and then we screened the stably transfected cell line with the SPAG6expression effectively down-regulated by RT-q PCR and Western blotting.The effects of SPAG6 knockdown on the proliferation,migration and invasion of SKOV-3 cells were explored by colony formation assay,wornd healing and Transwell invasion and migration assays.The expression and localization of cell cycle regulatory protein p27^kip1 in the control group and SPAG6 knockdown group were observed by immunofluorescence and Western blot.The p27^kip1 expression plasmid"p EGFPN2-p27^kip1"and SPAG6 expression plasmid"p En MCV-SPAG6"were co-transfected into HEK293T cells,and the localization of SPAG6 and p27^kip1 in the cells was observed by immunofluorescence,and the effect of SPAG6 overexpression on the localization of p27^kip1 was analyzed.The expression levels of PTEN,p-AKT,p-Thr157-p27^kip1,Caspase-9,cleaved Caspase-3 in the control and SPAG6 knockdown groups were examined by Western blot.The cells were treated with PI3K inhibitor,and the expression levels of SPAG6,PTEN,p-AKT,p-Thr157-p27^kip1,cleaved Caspase-3 in the cells were detected by Western blot.Results:The expression of SPAG6 m RNA in ovarian cancer cells SKOV-3 and OVCAR-3 was significantly higher than normal ovarian epithelial cells NOE-71,and the expression was the highest in SKOV-3 cells.Therefore,SKOV-3 cells were used for subsequent experiments.RT-q PCR and Western blot showed that"SPAG6- sh RNA3"and"SPAG6-sh RNA4"plasmids effectively inhibited the expression of SPAG6 in SKOV-3.Compared with the control,inhibition of SPAG6 expression could reduce the proliferation,the migration and invasion ability of SKOV-3 cells（P<0.01）.Immunofluorescence and Western blot results showed that p27^kip1was highly expressed in the cytoplasm of SKOV-3 cells.Inhibition of SPAG6 expression in SKOV-3 cells reduced the level of p27^kip1 in the cytosol and increased its levels in the nucleus.Meanwhile,p27^kip1 is highly expressed in the nucleus and hardly expressed in the cytoplasm of HEK293T cells.When p27^kip1 expression plasmid was co-transfected with the SPAG6 expression plasmid,p27^kip1 was expressed in the cytoplasm and the nucleus of HEK293T cells.Western blot showed that the expression of PTEN,Caspase-9 and cleaved Caspase-3 were increased and the expression of p-AKT and p-Thr157-p27^kip1were decreased in the"SPAG6-sh3"and"SPAG6-sh4"cells,compared with the control cells（P<0.01）.After the control cells and"SPAG6-sh4"cells were treated with LY294002,the expression level of SPAG6 did not change,but the expressions of PTEN and cleaved Caspase-3 were increased and the expressions of p-AKT and p-Thr157-p27^kip1 were decreased（P<0.05）.Conclusions:Low expression of SPAG6 can reduce the proliferation,growth,migration and invasion ability of ovarian cancer cells SKOV-3;SPAG6 can mediate the cytoplasmic localization of p27^kip1;SPAG6 can phosphorylate the p27^kip1 and promote apoptosis through the PTEN/PI3K/AKT signaling pathway to affect the proliferation of SKOV-3 cell.