Effects Of Phenytoin On The Expression Of Satb2 And Hoxa2 Gene In C57BL/6 Mouse Embryonic Craniofacial Tissue

Background and ObjectiveThe development of face comprises series of complicate events,including proliferation, migration and differentiation of neural crest cells,production of cellular matrix and programmed cell death.Till now,more and more genes have been known to play very significant roles in this process,such as:TGFα,TGFβ3,Msx1,IRF6,etc.Any disturbance of exogenous teratogens or abnormal expression of certain endogenous genes in this process is possible to result in craniofacial defects,and the most frequent defects are congenital cleft lip and cleft palate. Phenytoin is a major researched teratogen,but reports on the mechanism of cleft lip and cleft palate induced by phenytoin exist a lot of differences.There are mainly two views nowadays: One is due to the activation of prostaglandin H synthase(PHS) and reactively oxidative damage of DNA and embryonic protein;another is the fusion failure of palatal shelves caused by phenytoin inducing low expression of Hoxa2 mRNA.SATB2 gene has recently been found to be closely implicated in human face embryogenesis.Murine Satb2 gene is specifically expressed in maxillary prominences,frontonasal prominence and the medial edges of the developing palatal shelves.Satb2 gene can repress the expression of several Hox genes including Hoxa2,and this suggests that phenytoin and Satb2 gene may interact with each other in the development of cleft lip and cleft palate.Through real time PCR with Taqman probe technique,this experiment is to investigate the effects of phenytoin on Satb2 and Hoxa2 gene expression in craniofacial tissue of C57BL/6 mouse embyos,to explore the mechanism of phenytoin in the development of cleft lip and cleft palate,and to provide reference and proof for finding prophylaxis and adjunctive treatment of phenytoin inducing cleft lip and cleft palate.Method1.30 pregnant C57BL/6 mice were assigned randomly to phenytoin group(PHT) and normal saline group(NS),and then they were divided into three subgroups according to the conception time,namely GD11.5(Gestation Date,GD),GD12.5 and GD13.5 group.Each group consisted of five mice.2.Mice in PHT group were injected intraperitoneally with phenytoin on gestation day 10 (65 mg/kg,diluted in normal saline).Control group received normal saline(10 ml/kg) in the same manner.The animals were sacrificed by cervical dislocation at gestation day 11.5,12.5 and 13.5 respectively,and we isolated craniofacial tissue of mouse embryos under anatomical microscope.3.Total RNA was isolated by guanidinum thiocyanate-phenol-chloroform method,The integrity and purity of RNA were identified by agarose gel electrophoresis and UV spectrophotometric method.4.RNA samples were treated with DNase,and then reversely transcribed to the first chain cDNA.5.The change of Satb2 and Hoxa2 expression was examined by real time PCR with Taqman probe technique.Result1.Both Satb2 gene and Hoxa2 gene expressed in craniofacial tissue of normal mouse embryos dating from 11.5 to 13.5.The relative expression amount of Satb2 gene was 14.022, 40.33 and 66.412 respectively;and the relative expression amount of Hoxa2 gene was 1.682, 5.87 and 1.337 respectively.There was a significant difference in the expression amount of different time point.2.Compared with control group,the expression amount of Satb2 gene in corresponding time point decreased significantly in PHT group(P＜0.01);While the expression amount of Hoxa2 gene was higher than that in control group(P＜0.01).ConclusionSatb2 and Hoxa2 genes expressed in a key stage of mouse lip and palate embryogenesis, and this further testified that Satb2 and Hoxa2 genes were very important genes in mouse lip and palate embryogenesis.The expression amounts of Satb2 and Hoxa2 genes were significantly different at different time points.From mouse embryonic day 11.5 to 13.5,the expression amount of Satb2 gene in craniofacial tissue increased gradually;the expression amount of Hoxa2 gene grew firstly and then decreased.When the C57BL/6 pregnant mice exposed to phenytoin, the expression amount of Satb2 gene in craaiofacial tissue of mouse embryos decreased significantly,and the expression amount of Hoxa2 gene relatively increased.Phenytoin may induce cleft lip and cleft palate by regulating the expression of Satb2 and Hoxa2 genes.