Cisplatin induces apoptosis of ovarian granulosa cells by regulating FTO expression
Objective:To observe the expression of fat mass and obesity-associated protein(FTO)and the level of apoptosis in human ovarian granulocytes treated with cisplatin for investigating the effect of FTO gene on granulocyte apoptosis after chemotherapy.Methods:KGN cells were treated with cisplatin by concentration at 0,5,10,15 and 20 μmol/L for 24,48 and 72 h,respectively.The changes in cell proliferation ability were analyzed using CCK-8 method,the level of apoptosis was detected by flow cytometry after 48 hours of treatment.Western blot was used to measure the expression levels of FTO,Bcl-2 and Bax proteins in the cells.KGN cells were transfected with small interfering RNA(siRNA)targeting the FTO gene(siFTO group),and a cell control group(Control group,only containing liposomes)and a siRNA control group(siNC group,transfected with negative control sequences)were established.KGN cells were then transfected with FTO eukaryotic expression plasmids(FTO group),and a cell control group(Control group,only containing liposomes)and an empty vector control group(Vector group,transfected with empty vectors)were established.After transfection,cells were simultaneously treated with 5 μmol/L cisplatin for 48 h.finally,the expression levels of FTO,Bcl-2,and Bax in each group were detected using qRT-PCR and Western blot.Results:The proliferation ability of KGN cells gradually decreased in each treatment time group with added concentration of cisplatin(P＜0.05),and the level of apoptosis was gradually increased with cisplatin concentration addition(P＜0.05),yet FTO expression was gradually downregulated(P＜0.05).The relative expression levels of Bcl-2 decreased and Bax were increased after downregulating FTO using siRNA(both P＜0.001),and the relative expression levels of Bcl-2 was increased(P＜0.01),yet Bax was decreased after upregulating FTO using eukaryotic expression plasmids(P＜0.001).Conclusion:Cisplatin can downregulate the expression level of FTO in KGN cells and promote apoptosis of KGN cells by inhibiting the expression of apoptotic protein Bcl-2 and upregulating the expression of proapoptotic protein Bax.Chemotherapy may promote apoptosis of ovarian granulosa cells through FTO expression,leading to premature ovarian failure.
premature ovarian failurefat mass and obesity-associated proteinovarian granulosa cellssmall interfering RNAcell apoptosis
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