Study On The Relationships Of IFT88 In Mouse EPM Cells Cilia And Expression Of Sonic Hedgehog Signaling And Disassembly Of Primary Ciliary

Objective: Using RNAi technology to knock down Ift88,the intraflagellate transport system(IFT)related component of primary cilia on the embryonic palatal mesenchymal(EPM)cells,to explore the relationship between primary cilia and Sonic Hedgehog(Shh)signal transduction in EPM cells and the role of IFT88 in ciliary disassembly.Methods: C57BL/6J mouse embryonic palatal mesenchymal cells were cultured in vitro for 13.5 days and identified.The cultured cells were divided into three groups: Lentivirus transfection knock down Ift88 cells in the experimental group,conventional culture cells in the blank control group,and empty vector lentivirus transfection cells in the negative control group.Experiments 1:The above three groups of cells were cultured with secretory Shh protein for 24 hours,then the protein was extracted.The Shh signaling pathway related factors Ptch1,Smo,Gli3 and Cyclin D1 were detected and compared by Western Blots,and comparing Gli3A/Gli3 R ratio change.At the same time,the three groups of cell ciliary disassembly related proteins Aurora A,phosphorylated Aurora A and HEF1 were compared;Experiment 2:Cells in the experimental group were divided into two groups,without secretory Shh protein(group A)and with secretory Shh protein(group B).The ratio of Gli3A/Gli3 R and the expressions of Cyclin D1 were detected and compared,and analyzed statistically.Results: Experiment 1:There was no significant difference in Smo and Ptch1 expression among groups.The ratio of Gli3A/Gli3 R increased in the experimental group compared with the blank control group(P<0.05).The expression of Cyclin D1 in the experimental group was higher than that in the blank control group(P<0.05).The expression of HEF1 in the experimental group was lower than that in the blank control group(P<0.05).The expression of Aurora A in the experimental group was lower than that in the blank control group(P<0.05).Phosphorylated Aurora A showed no significant difference between the experimental group and the blank control group and the negative control group.Experiment 2:The expression of Cyclin D1 and Gli3A/Gli3 R ratio in experimental group B were higher than those in experimental group A(P<0.05).Conclusion: The results of this study indicate that the knock down of Ift88 affects the expression of Shh signaling pathway-related components and ciliary disassembl-related proteins.Shh signal maybe involved in non-canonical signal transduction in mouse EPM cells,and IFT88 may affect cilia regulators HEF1 and Aurora A,which further affect cilia stability.