| ObjectivesIn this study,a cleft palate model was established by exposure to all-trans retinoic acid(atRA)during pregnancy to explore the role of long non-coding RNA(lncRNA)Meg3 in cleft palate(CP)induced by excessive retinoic acid and the potential influencing mechanism.MethodsThis research used C57BL/6N inbred mice as the research object.All SPF animals were received at 6～8 weeks of age and adaptability held for 1 week before mating in the barrier condition.Two females were housed overnight with one male at 8:00 p.m.and checked at the 8:00 a.m.the next day for the presence of a vaginal plug,denoted as the gestation day 0(GDO)and record the weight.The body weight of pregnant rats was recorded again at 8:00 a.m.of GD10,and the body weight gain during this period was greater than or equal to 2.00 grams,which was considered as true pregnancy.On GD10,pregnant mice were randomly divided into experimental group(n=45)and control group(n=45)according to the principle of similar body weight.The experimental group was intragastrically given 100 mg/kg atRA solution dissolved in the corn oil at 10:00 a.m.,while the control group was only given an equal volume of corn oil solution.The mice were anesthetized and sacrificed on GD13,GD14,GD15,and collected the fetuses’ head respectively.HE staining was performed after paraffin embedding to observed the palatal development dynamics of GD13～GD15 fetal mice.The expression of lncRNA Meg3gene in mouse embryonic mesenchymal(MEPM)was observed by RT-qPCR and fluorescence in situ hybridization(FISH)assay.The expression of PCNA in MEPM was detected by RT-qPCR and the BrdU immunofluorescence assay were used to detected the proliferation of MEPM in the atRA group and the control group.On GD14,collected the atRA-treatment group and the control group embryonic palatal tissues,separated MEPM and extracted the total DNA respectively.The methylation status of Meg3 gene promoter in the control group and the atRA group on GD14 was evaluated by sulfite sequencing method.In addition,the expression of Smads signal proteins(p-Smad2,Smad2,Smad4,and Smad7)on GD14 was detected by Western blot.The effect of atRA on the binding of Meg3 gene to Smad2 protein on GD14 was investigated by immunoprecipitation assay.The statistical results were analyzed using SPSS 21.0,and the test levels was α=0.05.Results1.Palatal development of fetal mice:The results showed that the incidence of CP was 97.2%in the atRA group,and no other deformities occurred.GD13～GD15,HE staining showed that fetal mice in atRA group showed a cleft palate phenotype with delayed elevation of bilateral palatal process and incomplete contact fusion.The palate of the control group developed normally.2.The effect of atRA on the proliferation of MEPM cells:BrdU showed that compared with the control group,the number of BrdU positive cells in atRA-treatment group during GD13～GD15 was significantly reduced(P<0.05),while the results of GD15 were just opposite.In addition,RT-qPCR results also showed that the expression of PCNA in MEPM cells treated with atRA was significantly decreased(P<0.05).3.Expression of Meg3 in the palatal mesenchymal:RT-qPCR results showed that the expression of lncRNA Meg3 in the palatal mesenchymal during GD13～GD15 was significantly increased in the atRA group compared with the control group(P<0.05).In addition,FISH test also showed that Meg3 gene was mainly located in the nucleus of MEPM on GD14,which also confirmed that Meg3 expression was up-regulated in the atRA exposure group.4.Meg3 promoter methylation:Sulfite sequencing analysis showed that there was no difference in the overall methylation status of the four Meg3 gene promoter regions between the control group and the atRA group on GD14(P>0.05),while the individual CpG sites were significantly demethylated in the atRA-treated group(P<0.05).5.Expression of Smads signaling molecules:Western blot results showed that compared with the control group,the protein expression of p-Smad2 and Smad4 in atRA group were significantly decreased,and the protein expression of Smad7 was significantly increased(P<0.05),while Smad2 protein expression had no difference between the control group and the atRA group(P<0.05).6.Interaction between Meg3 and Smad2:RIP experiment results showed that Smad2 can directly interact with Meg3 in MEPM after atRA treatment(P<0.05).Conclusions1.AtRA could increase Meg3 expression in MEPM cells.2.The increased expression of Meg3 could be associated with the inhibition of MEPM cell proliferation.3.AtRA treatment could be associated with demethylation of the CpG sites in the Meg3 gene promoter.4.Smad2 could be the target of Meg3 gene in the context of atRA-induced cleft palate. |
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