The Study Of CPLANE1 Maintain The Number Of Primary Cilia By Regulating Hh Signal Pathway

Objective:Primary cilia is the transduction sites of many important signal pathways including the Hh signal pathway.The abnormality of the Hh signaling pathway is closely related to birth defects.Therefore,it is important to study the relationship between the development of primary cilia and the Hh signaling pathway.In our pervious study,we found that mutations in the CPLANE1 gene can cause fetal malformations.Used siRNA to knock down the expression of CPLANE1 gene could reduce the number of primary cilia.Whether CPLANE1 can affect the number of primary cilia by regulating the Hh signaling pathway is still unclear.We aim to explore whether CPLANE1 gene can regulate the Hh signaling pathway to maintain the number of primary cilia.Methods:1)Construct a mouse model of CPLANE1 point mutation by CRISPR-Cas9 technology,and observed the development of mouse fertilized eggs;2)Western blot and indirect immunofluorescence were applied to confirm the expression of CPLANE1 in primary cilia;3)The sequence of si-CPLANE1 was transiently transfection into NIH/3T3 cells and the expression of CPLANE1 by qRT-PCR and Western blot;4)Observed the number of primary cilia were detected by indirect immunofluorescence;5)Western blot and qRT-PCR were used to detect the influence of CPLANE1 knock down on the expression of Shh,Smo,Ptchl and Glil in Hh signaling pathway;6)Knock down CPLANE1 and added the Hh activator SAG to detect the expression of Shh,Smo,Ptch1 and Glil and the number of primary cilia via Western blot,qRT-PCR and indirect immunofluorescence.Results:1)The proportion of blastocyst stage was significantly reduced after CPLANE1 point mutation;2)We confirmed that the mouse embryonic fibroblast cell line NIH/3T3 cells express the CPLANE1 as shown by Western blot and indirect immunofluorescence;3)The expression of CPLANE1 was confirmed to be knocked down through Western blot and qRT-PCR;4)The number of primary cilia would to be reduce by CPLANE1 knock down NIH/3T3 cell;5)Down-regulated the level of Shh,Smo and Glil and up-regulated the level of Ptch1 in CPLANE1 knock down NIH/3T3 cells were confirmed by Western blot and qRT-PCR;6)Inhibited Hh signal pathway in CPLANE1 knock down NIH/3T3 cells could be reverse by added Hh signaling pathway activator SAG,and the number of primary cilia returned to be normal as shown by confocal laser scanning microscope.Conclusion:1)CPLANE1 expresses in NIH/3T3 cells.CPLANE1 knock down leads to decrease in the number of primary cilia.2)CPLANE1 can maintain the number of primary cilia via activating Hh signaling pathway.