Study On The Effect Of RNA Methylation On Male Reproductive Injury Induced By Bisphenol S

BackgroundIn recent years,the hazard to human health of the plasticizer bisphenol A（BPA）,which is widely used in the world,has attracted great attention and has been banned in many countries.At present,emerging plasticizers such as BPS,BPF,BPAF have begun to be used as substitutes in the market.Among them,Bisphenol S（BPS）is the most widely used,and its exposure level in the population and the detection rate is also higher.Studies have found that exposure to BPS in animal and human has many adverse effects on the reproductive system.However,the specific mechanism is not clear.Our previous study showed that exposure to environmental toxicants altered the overall level of N6-methyladenosine RNA methylation and the expression of RNA methylation-regulated genes.As very important RNA demethylase and binding protein,FTO and YTHDF1 are closely related to various biological processes of reproductive development.Combined with the latest literature reports,we intend to explore whether FTO and YTHDF1 potentially affects male reproductive function by regulating the dynamic balance of downstream key molecules in the process of BPS exposure-induced germ cell damage in mice.To this end,this study used bioinformatics analysis combined with mouse testicular spermatocytes GC-2 and Leydig cells TM3 cells in the mouse reproductive system to conduct experimental studies to explore the epigenetic regulation and its specific mechanism of RNA methylation mediated downstream regulatory pathway and key molecules in the process of male germ cells injury induced by BPS exposure.This study will provide new ideas for finding more sensitive molecular markers for male reproductive damage from the perspective of epigenetic regulation.Objectives1.To explore the possible pathways and genes of BPA and BPS leading to testis cell damage.2.To clarity the role and molecular mechanism of FTO gene in the process of male reproductive system cell injury induced by BPS through regulating apoptosis and cell cycle signaling pathway.3.To clarity the role and molecular mechanism of YTHDF1 gene in the process of male reproductive system cell injury induced by BPS through regulating apoptosis and cell cycle signaling pathway.Materials and Methods1.The genes related to male genitourinary toxicity caused by BPA and BPS toxicity were screened by CTD database.The gene sets were formed by the intersection of these genes,and the above gene sets were analyzed by GO function enrichment analysis,KEGG signal pathway enrichment analysis and visual analysis（Enrichment Map,cnetplot）in R language.2.Mouse spermatocyte cell line GC-2,Leydig cells TM3 and Sertoli cells TM4 were treated in the final concentration of 0μmol/L BPS（DMSO control group）,10^-9-10^-4mol/L for 72 h.The CCK-8 method was used to determine the viability.Mouse Leydig cells TM3were treated in the final concentration of 0μmol/L,20μmol/L,40μmol/L and 80μmol/L BPS for 72 h.The m RNA and protein expression levels of key genes were detected by q RT-PCR and Western blot.3.GC-2 and TM3 cells were treated with BPS at final concentrations of 0,20,40,80,160,320μmol/L and 0,20,40,and 80μmol/L,respectively,for 72 h.The cell morphology changes were observed under the microscope.The CCK-8 method was utilized to detect cell viability,and flow cytometry was used to detect cell apoptosis and cycle changes.Colorimetric method was used to detect the global level of m6A RNA methylation.Western blot was used to detect the protein expression levels of METTL3,FTO,YTHDF1,apoptosis and cell cycle-related molecules.A cell model with differential expression of FTO and YTHDF1 gene were established to analyze the effect of FTO on GC-2 cell and YTHDF1 on TM3 cell.RIP kit was used to detect the binding of YTHDF1 to key molecules of cell apoptosis and cell cycle.4.The statistical software used was SPSS 25.0 software.The results were the average of the three experimental results,and the data was expressed as the mean±standard deviation.The t test or one-way analysis of variance was used to compare the groups,and the differences between the control group and the experimental group were analyzed.P value<0.05,indicating a statistical difference.Results1.The possible pathways and genes associated with male reproductive system cell damage induced by BPA and BPS.GC-2,TM3 and TM4 cells were exposed to different doses of BPA and BPS.With the increase of exposure dose,the apoptosis rate increased significantly（P<0.05）.The enrichment analysis of related genes retrieved from CTD database combined with experimental studies found that the m RNA and protein expression levels of PI3K-AKT and HIF-1 signaling pathway related genes were changed（P<0.05）.The m RNA expression level of RNF130,a specific gene of male urinary system disease caused by BPS,was significantly increased（P<0.01）.2.The role and mechanism of FTO gene in the process of GC-2 cell injury induced by BPS.The cell survival rate was significantly decreased and the apoptosis rate was markedly increased at 160 and 320μmol/L of BPS（P<0.05）.The protein level of apoptosis-related molecule P53,P21,Caspase9 and Caspase3 increased significantly,and the protein level of BCL2 decreased significantly（P<0.05）.The protein levels of G1/S phase related molecules Cyclin D1,CDK4,and CDK2 were significantly down-regulated（P<0.05）.The overall content of RNA methylation was relatively increased（P<0.01）.The protein level of RNA demethylase FTO decreased sharply（P<0.05）.Compared with the control group,the viability of GC-2 cells was significantly decreased and the apoptosis rate was significantly increased.The protein level of P53 was significantly up-regulated and the protein level of BCL2 was significantly down-regulated after interfering with the expression of FTO（P<0.05）.3.The role and mechanism of YTHDF1 gene in the process of TM3 cell injury induced by BPS.With the increase of BPS exposure concentration,the survival rate of TM3 cells decreased and the apoptosis rate was markedly increased.The protein levels of apoptosis-related molecules Caspase9,Caspase3 and BAX increased significantly,and the protein level of BCL2 decreased significantly（P<0.05）.The cell cycle was blocked in G1/S phase,and the protein levels of G1/S phase related molecules CDK2,Cyclin E1were significantly down-regulated（P<0.05）.The overall level of RNA methylation was significantly decreased（P<0.0001）.The protein level of FTO was significantly increased,and the protein level of YTHDF1 was significantly decreased（P<0.05）.After overexpression of YTHDF1 gene,the apoptosis and cell cycle arrest of TM3 cells were inhibited,and the protein levels of its related molecules changed significantly（P<0.05）,while the m RNA expression levels had no significant changes.After the low expression of YTHDF1 gene,the apoptosis of TM3cells showed an upward trend,and the cell cycle was arrested in the G1/S phase.Their related molecular protein levels changed significantly（P<0.01）,while the m RNA expression levels did not change significantly.The m RNA binding of YTHDF1 protein to CDK2,Cyclin E1and BCL2 was significantly increased in TM3 cells overexpressing YTHDF1（P<0.01）.Compared with the 0μmol/L BPS group,TM3 cells with overexpression of YTHDF1 gene were then exposed to BPS.The relative growth rate of in the 80μmol/L BPS group was significantly changed（P<0.01）.Conclusions1.BPA is more toxic than BPS in GC-2 cells and TM3 cells.While in TM4 cells,BPA is comparable to that of BPS.PI3K-AKT and HIF-1 signaling pathway may play an important role in the growth and apoptosis of TM3 cells exposed to BPS.The results showed that BPS still showed a certain male reproductive toxicity effect.2.It is found that FTO gene is involved in the process of male reproductive toxicity caused by BPS,which may be through RNA methylation modification to regulate the expression of P53-P21 and BCL2 mitochondrial pathways and affect their translation levels.RNA demethylase FTO can be used as a potential biomarker for male reproductive injury induced by BPS.3.It is found that YTHDF1 gene is involved in the process of male reproductive toxicity caused by BPS,which may be through RNA methylation modification to regulate the mitochondrial pathway of BCL2 and the expression of CDK2-Cyclin E1 to affect its translation level.RNA binding protein YTHDF1 may serve as a potential biomarker for male reproductive injury induced by BPS.