Study On The Mechanism Of IFT122 Regulating The Cell Proliferation Of Mouse Embryo Palatal Mesenchymal Through The Primary Cilia-mediated Shh Signaling Pathway

Objective: This study intends to explore the relationship between IFT122,a key component of m EPMCs primary cilia intraflagellar transport(IFT),and Shh signaling pathway through in vitro experiments,as well as the mechanism of IFT122 regulating the proliferation of m EPMCs,in an attempt to preliminarily clarify the role of m EPMCs primary cilia and Shh signaling pathway in palate development,so as to provide an early theoretical basis for the prevention and treatment of cleft palate.Methods: C57BL/6J mouse embryonic palatal tissues of ED13.5 were obtained in vitro,and primary m EPMCs were obtained by enzyme digestion combined with mechanical separation,and the primary m EPMCs were identified by immunocytochemistry.The expression of IFT122 was down-regulated by RNAi technology,and the efficiency of transfection was verified by real-time PCR and western blot technology.(1)m EPMCs was then divided into the untreated Control Group and the IFT122-Kd Group,which down-regulated the expression of IFT122.AA-Tub(RFP)was labeled by Immunofluorescence technology to observe the formation of primary cilia,and Smo(GFP)was labeled to observe the change of fluorescence density;Real-time PCR and western blot technology detected the expression changes of Smo and Gli3,key receptor and transcription factor in the Shh signaling pathway;also the expression changes of PCNA,marker of cell proliferation,and the cell cycle factor Cyclin D1;and the quantity change of m EPMCs was detected by CCK-8 method.(2)SAG was added in IFT122-Kd Group,and m EPMCs was divided into Control Group,IFT122-Kd Group and IFT122-Kd+SAG Group.The co-localization of Smo and primary cilia was observed using immunofluorescence Co-Localization technique to mark AA-Tub(RFP)and Smo(GFP)simultaneously;The expressions of Smo,Gli3,PCNA and Cyclin D1 were detected by Real-time PCR and western blot technology;The quantity change of m EPMCs was detected by CCK-8 method.Results: Immunocytochemistry results showed positive expression of Vim and negative expression of P-CK in m EPMCs.(1)Immunofluorescence results showed that the incidence and length of primary cilia in IFT122-Kd Group decreased,and the fluorescence density of Smo decreased;Real-time PCR and western blot results showed that the expressions of IFT122,Smo,Gli3,PCNA and Cyclin D1 in the IFT122-Kd Group were lower than those in the Control Group;CCK-8 results showed that the growth rate of m EPMCs in IFT122-Kd Group slowed down and the number began to decrease at 96 h compared with before.(2)Immunofluorescence Co-Localization results showed that the addition of SAG promoted the Smo to enter the primary cilia in large quantities;Real-time PCR and western blot results showed that the expressions of Smo,Gli3,PCNA and Cyclin D1 in the IFT122-Kd + SAG Group were higher than those in the IFT122-Kd group,but still less than that in the Control Group;CCK-8 results showed that the number of m EPMCs in IFT122-Kd +SAG Group was higher than that in IFT122-Kd group,but still less than that in the Control Group.Conclusion:(1)IFT122-Kd results in defective primary cilia growth of m EPMCs;(2)IFT122 can regulate the proliferation of m EPMCs through Shh signaling pathway;(3)The conduction of Shh signaling pathway in m EPMCs depends on primary cilia.