Influence Of MID1 And IRF6,genes Associated With NSCLP,on Cell Proliferation,apoptosis,migration And EMT

Craniofacial development depends on the timely and spatiouly regulated cell proliferation,migration to ensure the convergence of the five facial primordiums in the midline.The subsequent apoptosis accompanied with the epithelial-mesenchymal transition(EMT)lead to the proper fusion of these embryonic prominences and disruption in any of these processes may result in cleft lip/palate(CLP).CLP can be the only manifestion in isolated cases or be presented as part of the malformations in a a syndrome.Multiple genes have been implied in regulating craniofacial development,amongst them,MID1,MID2,IRF6,MSX1,PTCH1,PVRL1 and JAG2,mutations in which mutations have been reported in both syndromatic and non-syndromatic CLP.Therefore,these genes are likely to be involved in regulating craniofacial development.However,the detailed functions of these genes and the molecular etiology of CLP are still not clear.Objective: the purpose of this research is to study the molecular functions of IRF6 and MID1 and to investigate their influence on cell proliferation,migration,apoptosis and EMT.Methods: 1?In this study,IRF6 and MID1 eukaryotic expression vectors were constructed using RT-PCR.The specific IRF6 and MID1 si RNA was synthetized by Sigma.2?Quantitative real-time PCR and Western blot were performed to detect the levels of m RNA and proteins after IRF6 and MID1 gene silencing in 293 T cells.3?The migration of the transfected cells were assessed using wound healing assay.4?The viability and and apoptosis of the transfected cells were detected by the CCK8 assay and flow cytometry,respectively.5?The morphology of the transfected cells was examined by the inverted fluorescence microscope.and the expression of E-cadherin and vimentin were monitored using Western-blot,Quantitative real-time PCR and immunofluorescence.Results: 1?After being transfected into 293 T cells,IRF6 and MID1 expression were significantly down-regulated by IRF6-si RNA-869(P<0.01)and MID1-si RNA-1678(P<0.001).2?In cells with IRF6 silencing,E-cadherin and Vimentin expression were both up-regulation(P<0.05,P<0.01).E-cadherin expression was down-regulation(P<0.05)while the Vimentin expression up-regulation(P<0.01)when the MID1 gene silencing.3?The migration ability and the viability of 293 T cells transfected with two si RNAs were significantly enhanced(P<0.001).4?Flow cytometry assay exhibited the apoptosis rate of transfected 293 T cells had no defference to the normal cell(P>0.05).In other wise,CCK8 analysis showed that the viability of 293 T cells with IRF6 and MID1 gene silencing were obviously inhibitory(P<0.001,P<0.001).5 ? The morphology of the transfected cells of IRF6-si RNA-869 and MID1-si RNA-1678 changed the adherens,tight junctions into no junctions.In addition,different from the multilateral and irregular forms,cells were spindle,even the original tentacles also disappear.Conclusion: 1?MID1 silence inhibited cell proliferation.In contrast MID1 can promote cell proliferation;2?E-cadherin was down-regulated after MID1 gene expression.MID1 can inhibit the EMT;3? IRF6 silence resulted in promotion of cell proliferation.In contrast that IRF6 can repress cell proliferation;4?E-cadherin was up-regulated after IRF6 gene silence.IRF6 can activate the EMT.5?Both MID1 and IRF6 have no effect on cell apoptosis,and they also can inhibit the cell migration.