Function Of Dishevelled 2 On Ciliogenesis Affected By Different Fluid Shear Stress

Objective:To explore the effects of different fluid shear stress on ciliogenesis and the role of Dvl2 in this process by constructing Dishevelled2(Dvl2)lentiviral interference and overexpression vectors.Methods:(1)A fluid dynamic cell model with adjustable shear stress was constructed in vitro,and the hUVECs were divided into 3 treatment groups:static culture group(Static,STA),Disturbed Flow(DF),and Unidirectional Shear Stress(USS).ciliogenesis was detected using immunofluorescence technology and the expression of primary cilia related genes and proteins were also detected by real-time PCR and western blot technology from the molecular and protein levels.(2)Intracellular location of Dvl2 andγ-tubulin was observed by immunofluorescence co-localization,and their intracellular interaction was verified by co-immunoprecipitation.The effect of different FSS on the relative expression of Dvl2 mRNA was detected by real-time PCR.(3)Lentiviral vectors interferes with the expression level of Dvl2 were constructed and were screened for stable transfectant cell lines,and they were divided into 4 groups:Bc(Blank control),Nc(Negative control),sh-Dvl2(RNAi interference group),and OE-Dvl2(overexpression group).In each group,virus infection efficiency and the expression of Dvl2 was detected.(4)Each group was loaded with DF and USS respectively,and primary ciliogenesis as well as primary cilia related genes and proteins were detected using immunofluorescence technique and real-time PCR and western blot respectively.Results:(1)A tunable fluid dynamic in vitro cell model was successfully constructed.Immunofluorescence technique showed that compared with STA group,The expression ofγ-tubulin was significantly increased(P<0.01)under the effect of DF,and significantly decreased under the effect of USS(P<0.01).Real-time PCR showed that compared with STA group,under the effect of DF,the relative expression of IFT88mRNA was significantly decreased(P<0.01)and the relative expression of Bbs8 mRNA was significantly increased(P<0.01).the relative expression ofγ-tubulin mRNA was significantly increased(P<0.05),while the FSS change to USS,the relative expression of IFT88 mRNA was significantly increased(P<0.05)and the relative expression of Bbs8mRNA was significantly decreased(P<0.01).the relative expression ofγ-tubulin mRNA was significantly decreased(P<0.05).The results of western blot had the same trend with real-time PCR,and all had statistically significant differences.(2)Immunofluorescence co-localization technique showed that Dvl2 localized toγ-tubulin,and co-immunoprecipitation showed that there was an intracellular interaction between Dvl2,Bbs8 andγ-tubulin.Compared with the STA group,the relative expression of Dvl2mRNA in group DF decreased significantly(P<0.01),while the relative expression of Dvl2 mRNA in group USS increased significantly(P<0.05).(3)Successful construction of lentivirus Nc(HBLV-GFP-PURO),sh-Dvl2(HBLV-h-Dvl2-GFP-PURO shRNA),OE-Dvl2(HBLV-h-Dvl2-GFP-PURO),virus titer>1x10~8 TU/mL,cell infection rate>98.6%;Real-time PCR showed that compared with Bc group,Dvl2 mRNA relative expression in Nc group had no significant difference(P>0.05),the relative expression of Dvl2 mRNA significantly reduced(P<0.01)in sh-Dvl2 group,the relative expression of Dvl2 mRNA in OE-Dvl2 group was significantly increased(P<0.01).The western blot results had the same trend with real-time PCR,and all had statistically significant differences.The cell line was successfully constructed.(4)The results of immunofluorescence showed that there was no significant difference in the expression ofγ-tubulin between the Nc group and the Bc group(P>0.05).The expression ofγ-tubulin was significantly increased in the sh-Dvl2 group(P<0.01).The expression ofγ-tubulin was significantly decreased in the OE-Dvl2 group(P<0.05).Under the USS the localization ofγ-tubulin had the same tendency with DF,and all had statistically significant differences.Real-time PCR showed that under the effect of DF.the relative expression levels of IFT88,Bbs8 andγ-tubulin mRNA in Nc group were not significantly different(P>0.05)compared with Bc group.The relative expression of IFT88 mRNA in sh group was significantly decrease(P<0.05),and the relative expression of Bbs8,γ-tubulin mRNA was significantly increased(P<0.05)in sh-Dvl2 group.The relative expression of IFT88 mRNA was significantly increased in OE-Dvl2 group(P<0.01),and the relative expression of Bbs8,γ-tubulin mRNA was significantly decreased(P<0.01),When USS was used,the relative expression of mRNA had the same trend with DF,and all had statistically significant differences.The results of Western blot showed that when under the DF,compared with the Bc group,There was no significant difference in the expression of IFT88,Bbs8,γ-tubulin protein(P>0.05)in Nc group.The expression of IFT88 protein was significantly decreased(P<0.01),and the expression of Bbs8,γ-tubulin protein was significantly increased(P<0.05)in sh-Dvl2 group,The expression of IFT88 protein was significantly increased(P<0.01),and the expression of Bbs8,γ-tubulin protein was significantly decreased(P<0.05)in OE-Dvl2 group,the protein expression level of USS have the same trendwith the DF effect,and all have statistical differences.Conclusions:Primary ciliogenesis could be promoted by DF,and inhabited by USS..Interaction of Dvl2 with ciliary basal body and tubulin in the presence of intracellular proteins.Primary ciligenesis could also be inhabit by Dvl2 overexpression,however,that was occurred by silencing Dvl2.Consequently,FSS influences the endothelial primary ciliogenesis through regulating the expression of Dvl2.