Role and mechanism of RNA demethylase FTO in apoptosis of GC-2 cells induced by bisphenol S

Abstract

Objective To explore the role and mechanism of RNA demethylase FTO in the apoptosis of mouse spermatocyte GC-2 cells induced by bisphenol S (BPS). Methods GC-2 cells were treated with BPS at a final concentration of 0 (DMSO control group), 20, 40, 80, 160, and 320 μmol/L for 72 h. The cell morphology changes were observed with microscopy. CCK-8 assay was utilized to detect cell viability, and flow cytometry was used to detect cell apoptosis and cycle changes. Colorimetric assay was adopted to measure the global level of m6A RNA methylation. Western blotting was employed to detect the protein levels of RNA methyltransferase METTL3, RNA demethylase FTO, RNA binding protein, YTHDF1, apoptosis-related molecules P53, P21, BCL2, Caspase9, Caspase3, BAX and cell cycle-related molecules CyclinD1, CDK4, CDK2. After FTO gene interference cell model was established, CCK-8 assay was used to measure cell survival and flow cytometry to detect cell apoptosis. The protein levels of FTO, P53, BCL2 and BAX were detected by Western blotting. Results Compared with the control group, the number of GC-2 cells was significantly reduced and more vacuole cells appeared in the BPS exposure group. The cell survival rate was significantly decreased and the apoptotic rate was markedly increased at 160 and 320 μmol/L of BPS (P < 0.05). The protein levels of apoptosis-related molecules P53, P21, Caspase9 and Caspase3 were increased significantly, and that of BCL2 was decreased significantly (P < 0.05). When the BPS concentration was 80, 160 and 320 μmol/L, cell cycle was arrested at G1/S phase (P < 0.05). The protein levels of G1/S phase related molecules CyclinD1, CDK4, and CDK2 were significantly down-regulated (P < 0.05). When the dose of BPS increased to 80~320 μmol/L, the global RNA methylation level was relatively increased (P < 0.01). The protein levels of RNA methyltransferase METTL3 and RNA binding protein YTHDF1 did not change obviously, while that of RNA demethylase FTO decreased sharply (P < 0.05). Compared with the control group, after interference with FTO expression, the number and viability of GC-2 cells were significantly reduced, and the apoptotic rate was notably increased (P < 0.05). The protein level of P53 was remarkably up-regulated, and that of BCL2 down-regulated when FTO expression was knocked down (P < 0.05). Conclusion RNA demethylase FTO may play an important role in BPS-induced apoptosis of GC-2 cells through regulating P53-P21 and BCL2 mitochondrial pathway

Keywords

• bisphenol s
• mouse spermatocytes
• rna demethylase fto
• apoptosis