The Effect Of ICK On Hedgehog Signaling Pathway And Differentiation In Preadipocytes

BackgroundPrimary cilia are widely existing on the surface of various cells as organelles,which are formed and maintained by the intraflagellar transport IFT.Primary cilia achieve two-way signal transmission through IFT.When preadipocytes are in the stage of growth stagnation,primary cilia are already formed.And they perform changes during the process of differentiation,which is of great significance to differentiation.In vertebrates,the maintenance of the Hedgehog signaling pathway depends on the primary cilia.Primary cilia provide a structural and functional compartment that allows the Hh signaling pathway to respond.The Hedgehog signaling pathway is associated with differentiation.Intestinal cell kinase ICK is located at the base of cilia and regulates the length of primary cilia in mammals.Previous studies have shown that when ICK is mutated,ICK's dysfunction causes structural damage of cilia.The effect of ICK mutations on the Hh signaling pathway depends not only on specific tissue and cell environments,but also on specific stages of growth and development.This study aims to discover the effect of ICK on Hedgehog signaling pathway and differentiation in preadipocyte,which may be mediated by cilia.Objective1.To observe the localization and dynamic changes of primary cilia in 3T3-L1 preadipocytes during the differentiation.2.To discover the temporal expression of ICK and the downstream genes of Hedgehog pathways during the differentiation of 3T3-L1 preadipocytes.3.To verify the effect of inhibition and activation of Hedgehog signaling pathway on differentiation of 3T3-L1 preadipocytes.4.To study the effect of ICK-shRNA on primary cilia,Hedgehog signaling pathway and differentiation of 3T3-L1 preadipocytes.Methods1.Acetylated ?-tubulin and ARL13B immunofluorescence staining of primary cilia were performed when 3T3-L1 preadipocytes were cultured for d0,d2,d4,d6 and d8 after differentiation.The location of primary cilia was observed under Confocal Laser Scanning Microscope,and the length of primary cilia was measured and analyzed.2.Total RNA was extracted from d0,d2,d4,d6 and d8 of 3T3-L1 cells after differentiation,and mRNA expressions of ICK,Glil and Gli2 were detected by qPCR.3.The Hh signaling pathway inhibitor Cy and the agonist SAG were used to intervene the cells,and a control group was set up.After 8 days of differentiation,cells were collected.mRNA expression of Hh signaling pathway marker genes Gli1,Gli2,Ptch1 and lipid-forming marker genes PPARy,CEBP? in each group was detected by qPCR.Oil red O staining was used to observe the formation of lipid droplets in 3T3-11 cells of each group.4.Cells were divided into three groups:ICK-shRNA,Vector and Mock,and all were induced to differentiate.Immunofluorescence staining of primary cilia were performed when 3T3-L1 preadipocytes were cultured for two days.The location of primary cilia was observed under Confocal Laser Scanning Microscope,and the length of induced primary cilia was measured and analyzed.Total RNA was extracted from each group at the differentiation of d0,d2 and d8,respectively to detect the mRNA expression of key genes in the Hh signaling pathway,including Glil,Gli2 and ptch1,as well as the mRNA expression of lipid-forming marker genes,including PPAR? and CEBP? at d8.Statistical analysisIBM SPSS Statistics 20 software was used for statistical analysis.Statistical values were expressed as mean standard deviation.Statistical curves and ICONS were made using Prism(graph pad).Each group of data was analyzed by unpaired two-tailed t test.P<0.05 was considered significant difference,and P<0.01 was considered extremely significant difference.Results1.Primary cilia exist in the process of adipocyte differentiation,and the primary cilia are the longest on d2 during differentiation,and then gradually become shorter.2.During the differentiation of preadipocytes,ICK has the lowest mRNA expression level on d2,while Gli1 and Gli2 both have the highest mRNA expression level on d2.3.Inhibition of the Hedgehog signaling pathway can promote differentiation,while activation of the Hedgehog signaling pathway can inhibit differentiation.4.Silencing ICK can prolong the length of primary cilia during preadipocytes differentiation,up-regulate the expression of Gli1,Gli2 and ptch1,and inhibit the differentiation of preadipocytes.ConclusionICK is a key regulator of Hedgehog signaling pathway and differentiation of preadipocytes.Silencing ICK prolongs the primary cilia of the preadipocytes during differentiation,activates the Hedgehog signaling pathway,and inhibits the differentiation of preadipocytes.ICK may be a new target for the prevention and treatment of metabolic diseases such as obesity.