The Mechanism Of Long Non-coding RNA MEG3 In TCDD Induced Fetal Cleft Palate

ObjectivesIn this study,the 2,3,4,7-tetrachlorodibenzo-p-dioxin(TCDD)fetal cleft palate model was established to explore the mechanism of long non-coding RNA MEG3 in the process of TCDD inducing fetal cleft palate.Thus providing scientific theoretical basis for the prevention of cleft palate.MethodsSPF healthy adult C57BL/6N mice were caged at about 8 o ’clock in the evening of the previous day with a male to female ratio of 3:1.At 8 o ’clock in the morning of the second day,the weight of female mice was recorded and the presence of milky white solid glue in the vagina of the mice was examined.If there was,it was marked as gestation day 0(GD0).The mice were weighed again at 8:00 a.m.on GD0,and if the weight increased by more than 2 g or more than GD0,they were classified into experimental group(n=31)and control group(n=31)by random number table method.The mice were orally injected with 64μg/kg TCDD and appropriate amount of corn oil,respectively.On GD13,GD14 and GD15,mice were sacrificed by cervical dislocation.Part of fetal rat heads were taken for embedding and section,and used for HE staining,in situ hybridization and Brd U fluorescence staining.The other part of palatal tissue was taken for molecular biological detection after homogenization.The effects of TCDD on MEG3 expression in mouse embryonic palate mesenchymal(MEPM)cells were investigated by real-time fluorescence quantitative PCR(q PCR)and in situ hybridization(FISH).Cell proliferation assay(Brd U)was used to detect the proliferation of MEPM cells at GD13,GD14 and GD15.Sulfite sequencing method was used to determine the methylation level of MEG3 gene promoters in the two groups on GD14.Meanwhile,western blotting was used to explore the effects of TCDD on the expression of proteins related to Smad pathway(p-Smad2,Smad2,Smad4 and Smad7).The effect of TCDD on MEG3 binding to TβRI protein was explored by RNA binding protein immunoprecipitation(RIP)experiment.IBM SPSS Statistics 25.0 software was used for statistical analysis of comparison between the two groups by independent sample t test.Bilateral P<0.05 was considered statistically significant.Results1.Influence of TCDD on palatal fusion: HE results showed that there was no significant change in palatal development between the two groups on GD13.At GD14,there was a relatively larger gap between the palatal plates in TCDD group.At GD15,the TCDD group showed obvious cleft palate due to the abnormal contact and fusion of bilateral palates,while the control group showed no cleft palate due to normal contact and fusion of bilateral palates.2.Influence of TCDD on MEG3 expression in MEPM: q PCR results showed that at GD13,GD14 and GD15,the relative expression level of MEG3 in THE TCDD group was significantly higher than that in the control group(P<0.05).Meanwhile,FISH experiment showed that MEG3 was mainly expressed and located in the nucleus of GD14.3.Effects of TCDD on proliferation of MEPM cells in critical palatal period:Brd U experiment showed that the positive cell rate of TCDD group was significantly lower than that of control group on GD13 and GD14(P<0.05),while the positive cell rate of TCDD group was significantly higher than that of control group on GD15(P<0.05).4.Effect of TCDD on methylation of MEG3 gene promoter: From the overall level,the overall methylation level of the TCDD group was slightly lower than that of the control group,with no statistical difference(P>0.05).However,from different loci,the 5th sites methylation levels of promoter 11 in TCDD group were significantly lower than those in the control group(P<0.05).5.Influence of TCDD on protein expression level of Smad pathway: On GD14,compared with the control group,the expression of Smad2 and p-Smad2 in MEPM cells of TCDD group was reduced(P<0.05),while the expression of Smad7 was increased(P<0.05),while there was no statistical difference in Smad4 expression(P>0.05).6.Influence of TCDD on the combination of MEG3 and TGFβR1: RIP experiment showed that MEG3 could bind to TβRI protein.Meanwhile,compared with the control group,TβRI protein in TCDD group was enriched with more MEG3(P>0.05).Conclusions1.During the development of cleft palate,TCDD may promote MEG3 expression by demethylation of partial MEG3 promoter sites through DNA methylation modification.2.TCDD may inhibit the proliferation of palatal mesenchymal cells in fetal rats by affecting MEG3 transcription level and inhibiting TGF-β/Smad signaling pathway through the targeted binding of MEG3 and TβRI,thus leading to the occurrence of cleft palate.