S-adenosylmethionine Depletion-mediated CYR61 M~6A Downregulation Is Involved In Arsenic-induced Trophoblast Invasiveness Inhibition And Fetal Growth Restriction

Intrauterine growth restriction(IUGR)refers to the failure of a fetus to reach its genetic growth potential.The main clinical manifestations are low birth weight infants(LBW infants)and small for gestational age infants(SGA infants).IUGR is an important risk factor for perinatal fetal death and adult metabolic disorders and schizophrenia.Previous studies have focused more on nutritional factors;however,Growing evidence suggests that harmful environmental factors are important risk factors for IUGR.Arsenic(As)is a kind of toxic metalloid ubiquitous in the environment.Globally,about 140million people drink arsenic-contaminated water.In recent years,the relationship between maternal arsenic levels and embryonic development has gradually attracted the attention of domestic and foreign experts.Epidemiological surveys and animal experiments have found that maternal arsenic exposure increases the risk of IUGR.However,the potential molecular mechanism of arsenic-induced IUGR is unclear.This study established an animal model of the effects of gestational arsenic exposure on placental and fetal development.To clarify the role of placental trophoblast invasion in arsenic-induced IUGR.Based on IUGR animal models,the protective effect of folic acid supplementation on arsenic-induced IUGR was further explored.A nested case-control study was used to compare maternal urinary arsenic levels and serum cysteine-rich angiogenic inducer(Cyr61)content in SGA and appropriate for gestational age infants(AGA infants).This study is meaningful for us to understand the etiological mechanism of IUGR,and preventing and controlling the occurrence of IUGR is important for reducing perinatal mortality.1.Effects of gestational arsenic exposure on placental and fetal developmentCD-1 pregnant mice were randomly divided into 4 groups,and were exposed to different concentrations of NaAsO₂ in drinking water from gestation day(GD)0 to GD18,CTRL group,As-L group(0.15mg/L NaAsO₂),As-M group(1.5mg/L NaAsO₂),As-H group(15mg/L NaAsO₂).All pregnant mice were scarified on GD18.The number of abortion,preterm delivery and resorption sites were counted for each litter.The results showed that compared with the CTRL group,the average number of resorption site in the As-M group and the As-H group was significantly increased;and the average number of dead fetus in the As-H group was significantly increased.The average body weight and crown-rump length of live fetuses in As-M and As-H groups were significantly lower than those in the control group.The placenta is an important organ that maintains the health of the mother and fetus.Further analysis found that placental weight and diameter were significantly reduced in the As-H group compared to the CTRL group.H?E staining showed that compared with the CTRL group,the ratio of cross-sectional thickness of labyrinth zone to trophoblast zone was reduced in As-M and As-H groups,and the percentage of the labyrinth zone area to the total placenta area was significantly reduced in the As-H group.By PAS staining and phenol concentrated sulfuric acid method,the results have shown that compared with the CTRL group,the As-M group and the As-H group had significantly higher trophoblast glycogen-positive cells and placental glycogen content.These results suggest that gestational arsenic exposure causes placenta abnormalities and IUGR.2.Effects of arsenic exposure on the migration and invasion of mouse placenta and human placental trophoblast cellsPlacental glycogen cells resemble human invasive extravillous trophoblast cells.Compare with the CTRL group,the protein expression of placental E-cadherin was markedly increased,while Vimentin,N-cadherin,MMP2 and MMP9 protein levels were significantly decreased.In vitro,the effect of arsenic exposure on the migration and invasion of human placental trophoblast cells was further investigated.After As treatment,the protein expression of Occuldin was significantly increased,while Vimentin,N-cadherin,MMP2and MMP9 protein levels were significantly decreased.These results suggest that arsenic exposure impairs placental development by inhibiting the migration and invasion of placental trophoblast cells.3.The regulatory effect of arsenic on Cyr61 m~6A modification in mouse placenta and human placental trophoblast cellsTo investigate the mechanism by which arsenic exposure inhibits the migration and invasion of mouse placenta and human placental trophoblast cells.The results showed that compared with the CTRL group,the As-H group had no significant changes in Tgf-?1 and Tgf-?2 mRNA levels;meanwhile,the p-Smad2/3 protein expression also had no significant changes.It is suggested that arsenic inhibits the migration and invasion of mouse placenta independent of Tgf-?signaling pathway.Further analysis showed that the Cyr61,an extracellular matrix protein marker,mRNA and protein levels in the As-H group were significantly lower than those in the CTRL group.Immunohistochemistry showed that Cyr61 was expressed in both the labyrinth and trophoblast of the placenta;however,the number of Cyr61 positive cells in the As-H group was lower than that in the CTRL group.In vitro experiments showed that Cyr61 mRNA and protein levels were significantly decreased after As treatment.Immunofluorescence results showed that the number of Cyr61 positive cells in human placental trophoblast cells was significantly reduced after As treatment.Abnormal mRNA stability affects mRNA levels and protein expression.The results showed that Cyr61 mRNA stability in human placental trophoblast cells was significantly decreased after As treatment.m~6A modification affects mRNA stability.After As treatment,total m~6A methylation level was increased;but,the m~6A level at the Cyr61modification site was significantly reduced.Moreover,after As treatment,the interaction between the insulin-like growth factor 2 mRNA-binding proteins 2(IGF2BP2),a m~6A reader protein,and Cyr61 mRNA was inhibited.These results suggest that arsenic downregulates Cyr61 m~6A modification and reduces the binding of Cyr61 mRNA to IGF2BP2,which in turn reduces Cyr61 mRNA stability,resulting in the blocking of placental trophoblast cell invasion and impairing placental development.4.The role of S-adenosylmethionine depletion-mediated Cyr61 m~6A downregulation in arsenic-induced trophoblast invasiveness inhibitionTo explore the mechanism of arsenic down-regulating Cyr61 m~6A modification.The results showed that the Mettl3,Mettl14,Wtap,Rbm15 and Rbm15b,five m~6A methyltransferases,mRNA levels were not significantly changed after As treatment;the Fto,a m~6A demethylase,mRNA level was not significantly changed,while the Alkbh5,also a m~6A demethylase,mRNA level was significantly decreased.Fortunately,the m~6A methyltransferase activity was significantly reduced after As treatment.S-adenosylmethionine(SAM)is not only a methyl donor for m~6A modification,but also an essential cofactor for the catalytic activity of methyltransferases.After As treatment,the m~6A methyltransferase activity was significantly reduced.Moreover,the intracellular SAM content was significantly reduced after As treatment.These results suggest that intracellular SAM depletion is one of the reasons that arsenic exposure leads to Cyr61m~6A modification reduction.Arsenic(+3)methyltransferase(As3MT)catalyzes the methylation of i As3+with SAM as a substrate.In vitro experiments showed that silencing of As3MT could reverse As-induced intracellular SAM content and m6A methyltransferase activity reduction.Meanwhile,Cyr61 mRNA stability and protein expression level were restored.The protein expressions of Vimentin,N-cadherin and MMP2 were restored.By Cell migration and invasion assays,As3MT silencing can alleviate As-evoked trophoblast invasiveness inhibition.In vitro SAM supplementation,the results showed that SAM supplementation could reverse As-induced SAM content and m~6A methyltransferase activity reduction.SAM supplementation could reverse the decrease in Cyr61 mRNA stability and protein expression level caused by As.SAM supplementation could restore the decreased expression levels of Vimentin,N-cadherin and MMP2 caused by As.SAM supplementation could alleviate As-evoked trophoblast invasiveness inhibition.5.Protective effect of folic acid supplementation on gestational arsenic exposure against placental and fetal developmentCD-1 pregnant mice were randomly divided into 4 groups,CTRL group,FA group,As group and As+FA group.Compared with the As group,the abortion rate and the average number of resorption site in the As+FA group were significantly decreased.In addition,the fetal weight and crown-rump length in the As+FA group were significantly increased compared with the As group.Compared with the As group,the placental diameter and weight in the As+FA group were significantly increased;moreover,the percentage of the labyrinth zone area to the total placenta area was significantly increased;and the ratio of cross-sectional thickness of labyrinth zone to trophoblast zone was also significantly increased.Compared with the As group,SAM content and m~6A methylase activity in the As+FA group were increased.Folic acid supplementation could reverse the As-induced decrease in Cyr61 and MMP2 protein expression levels.6.Case-control study:the correlation analysis between Cyr61 downregulation and arsenic-induced fetal growth restrictionFetuses were divided into AGA and SGA according to their neonatal weight.Using the maternal urinary As concentration as a marker of As exposure,the results showed that compared with the AGA group,the maternal urinary As content in the SGA group was significantly increased;however,the plasma Cyr61 content was significantly decreased.The correlation between maternal urinary As content and plasma Cyr61 content was further explored.In AGA group,maternal urinary As content was negatively correlated with plasma Cyr61 content(R Sq Linear=0.08,P<0.05);in SGA group,maternal urinary As content was negatively correlated with plasma Cyr61 content(R Sq Linear=0.26,P<0.05).Meanwhile,the expression of placenta Cyr61 was further detected by immunohistochemistry.Compared with the AGA group,the number of Cyr61 positive cells in the SGA group was significantly reduced.The protein expressions of placenta Vimentin,MMP2 and MMP9 were further detected.Compared with the AGA group,the protein expressions of Vimentin,MMP2 and MMP9 in the SGA group was decreased.Immunohistochemistry showed that the number of placenta Vimentin,MMP2 and MMP9positive cells in the SGA group were decreased compared with that of the AGA group.The present study further validated the correlation between Cyr61 downregulation and arsenic-induced IUGR in the case-control study.According to the above experimental results,the following conclusions can be drawn:firstly,gestational arsenic exposure inhibits the migration and invasion of trophoblast cells,which leads to abnormal placental development and fetal intrauterine growth restriction.Secondly,In vitro and in vivo experiments have shown that As exposure leads to insufficient Cyr61 m~6A modification,reducing Cyr61 mRNA stability and protein level,which is one of the reasons for trophoblast migration and invasion inhibition.Further analysis revealed that As-induced SAM depletion downregulates m~6A methyl donor and methyltransferase activities,which was one of the reasons for the insufficient Cyr61 m~6A modification.Then,folic acid supplementation could alleviate gestation arsenic evoked-abnormal placenta development and IUGR.Finally,in case-control study,there was a negative correlation between maternal plasma Cyr61 content and arsenic-induced IUGR.