Abstract:

Objective To discuss the effect of knock down of gene of kinesin family member 3B （KIF3B）， an important component of primary cilia （PC） of in mouse embryonic palatal mesenchymal on the autophagy level of cells （mEPMCs） cells， and to clarify its mechanism. Methods The mEPMCs from gestational day 14.5 C57BL/6J mice cultured in vitro were collected and divided into control group （administered normal saline）， empty lentivirus transfected cell group （sh-NC group） （administered lentivirus transfection）， KIF3B knockdown group （sh-KIF3B group） （administered KIF3B gene knockdown）， and KIF3B knockdown plus Smoothened receptor agonist （SAG） group （sh-KIF3B+SAG group） （administered KIF3B gene knockdown followed by SAG addition）， based on whether the KIF3B gene was knocked down and whether the SAG was used to activate the sonic hedgehog （Shh） signaling pathway and its downstream coreceptor Smo， with 5 rats in each group. Transmission electron microscope was used to observe the morphology and the number of autophagosomes/autolysosomes in the mEPMCs in various groups； Western blotting method was used to detect the expression levels of autophagy-related proteins Beclin-1 and p62， and the Shh signaling pathway proteins Shh and Smo in the mEPMCs in various groups. Results The transmission electron microscope observation results showed that compared with control group， the number of autophagosomes/autolysosomes in sh-KIF3B group was significantly increased （P<0.05）； compared with sh-KIF3B group， the number of autophagosomes/autolysosomes in the mEPMCs in sh-KIF3B+SAG group was significantly decreased （P<0.05）. The Western blotting results showed that compared with control group， the Beclin-1 protein expression level in the mEPMCs in sh-KIF3B group was significantly increased （P<0.05）， and the KIF3B， p62， Shh， and Smo protein expression levels were significantly decreased （P<0.01）； compared with sh-KIF3B group， the Shh， Smo， and p62 protein expression levels in the mEPMCs in sh-KIF3B+SAG group were significantly increased （P<0.01）， and the Beclin-1 protein expression level was significantly decreased （P<0.01）. Conclusion Knockdown of KIF3B gene can promote autophagy of the mEPMCs， and the mechanism may be related to its inhibition of the Shh signaling pathway.

Key words: Cleft lip and palate, Kinesin family member 3B, Primary cilia, Sonic hedgehog signaling pathway, Autophagy