| Primary cilium is an antennae-like structure protruding from the cell surface. Primary cilia regulate cell motility, act as a sensory organelle and organize diverse signaling pathways including Wnt, Hedgehog and PDGFR signaling pathways, which participate in many vital activities of organisms such as cell proliferation and differentiation. Defects in formation or function of primary cilia result in a range of cilia-related diseases. Adenomatous polyposis coli (APC) gene is an antitumor gene and its mutations are responsible for FAP and Gardner’s syndrome. Given that the common cilinical manifestations were present in cilia-related diseases and Gardner’s syndrome, APC may be involved in ciliary biogenesis. However, little is known about the association between APC and cilia.The main purpose of this study is to investigate the relationship between N-terminus of APC and cilia assembly. This thesis consists of three parts:Part one:Effects of APC and N-terminal fragments of APC on cilia formation. We examined levels of APC at different times during ciliary assembly of NIH3T3fibroblasts with western blotting and its localizations in cilia by IFM. We transfected NIH3T3fibroblasts with plasmids encoding full-length, N, M and C regions of APC fused to GFP, then observed the effects on cilia formation; Furthermore, we used NIH3T3and MDCK cells as models, focused on the effects of three N-terminal fragments of APC (APC-N1, APC-N2and APC-N3) on cilia formation. Results showed that APC was up-regulated during growth arrest and localized to the basal body region of primary cilia in NIH3T3fibroblasts. Expression of N-terminal fragments of APC (containing the amino acid sequence of1-1018,1019-2038and2039-2843) inhibited cilia assembly, among which APC-N2had the most significant influence campare to APC-N3and APC-N1.Part two:The mechanisms of N-terminal fragments of APC inhibiting cilia assembly. Above results suggested N-terminal fragments of APC inhibited cilia assembly. This part contains:1. Effect of three N-terminal fragments of APC on GSK3β.2. Lacalization of three N-terminal fragments of APC in cilia;3. Effect of over expression of APC-N1, APC-N2and APC-N3on y-tubulin and pericentrin;4. Effect of overexpression of APC-N1, APC-N2and APC-N3on acetylated tubulin;5. Effect of overexpression of APC-N1, APC-N2and APC-N3on cell cycle progression. The results showed that (1) N-terminal fragments of APC inhibited ciliogenesis through a pathway involving PI3K/AKT signalling, which were associated with GSK-3β inactivation.(2) Location of N-terminal fragments of APC on the basal body were different although they all inhibit ciliogenesis.(3) APC-N2and APC-N3affected the stability of y-tubulin during cilia assembly, which may give rise to abnormal ciliogenesis.(4) APC-N2inhibited cilia formation also through affecting acetylated tubulin levels.(5) N-terminal fragments of APC suppressed ciliary assembly not through disturbing cell cycle progression.Part three:Effect of N-terminal of APC on cilia-related signaling pathways, including Wnt, Hedgehog and PDGFR. We mainly examined the effect of three N-terminal fragments of APC on levels of the important components of Wnt, Hedgehog and PDGFR signaling pathway by western blotting. We discovered expression level of P-catenin was increased in Wnt pathway; Glil was increased, but Gli3-R was decreased in Hedgehog pathway; PDGFRa, p-Akt and p-ERK1/2were increased in PDGFR pathway. These results indicated that N-terminal fragments of APC actived cilia-related Wnt, Hedgehog and PDGFR signaling pathway. |
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