The Mechanism Of N-terminus Of Adenomatous Polyposis Coli Affecting Primary Cilia Assembly And Cell Adhesion

Adenomatous Polyposis Coli (APC), a multiple functional protein, participates in Wnt signaling、cell migration、cell adhesion、regulating cell polarity、chromosome separation, and so on. Cilium is an organelle projecting from cell surface. Defects in cilia structure and function were related to kinds of deseases. Cell adhesion includes cell-cell adhesion and cell-matrix adhesion, which play a key role in transfer of tumor. It has been reported that APC mutation could disturb cilia formation and cell adhesion, but the specific mechanism is unclear. Studies showed that a fragment of APC containing 1 to 448 amino acids (APC-N1) was to be retained in many APC mutation individuals. This fragment, containing a oligomerization domain, form complex with full-length APC and results in inaction of APC. In this research, we investigated the mechanism of APC-N1 affecting cilia formation and cell adhesion. This research consists of the following several parts:The first part:QT-PCR and western blotting were used to detect expressions of two members of Hedgehog signaling pathway (Ptchl and Gli1) in MDCK-GFP and MDCK-APC-N1 cells. The results showed Ptchl and Gli1 levels were significantly increased in MDCK-APC-N1 cells compared to that in MDCK-GFP cells, which suggested that overexpression of APC-N1 could activate Hedgehog signaling. Gli1 siRNA and Gli1 inhibitor GANT61 were used to detect the effects of Gli1 on cilia formation in MDCK-APC-N1 cells. The results showed downregulated Gli1 led to incresed cilia formation ratio, which suggested that APC-N1 inhibited cilia formation by activating Hedgehog signaling. Furthermore, β-catenin siRNA was used to reduce the expression of β-catenin and investigate the effect of P-catenin on Gli1. The results showed β-catenin siRNA resulted in downregulating Glil expression. Above results suggested that APC-N1 inhibited cilia formation by activing Hedgehog signaling mediated by β-catenin.The second part:QT-PCR was used to detect the effect of APC-N1 on expressions of AurA. The results showed AurA mRNA level was significantly increased. AurA siRNA and TSA were exployed to inhibit AurA and HDAC6, respectively. The results showed that reduced AurA or HDAC6 levels led to increased cilia formation radio, which suggested that APC-N1 induced cilia damage through AurA/HDAC6. RNA interference was used to reduce Gli1 and then detect the expressions of AurA and HDAC6. The results showed that reduced Gli1 resulted in downregulated AurA and HDAC6. Above results indicated that APC-N1 decreased cilia formation ratio by up-regulating AurA and HDAC6 levels, which were mediated by activing Hedgehog/Glil pathway.The third part:Cell aggregation assay、QT-PCR and western blotting were used to detect the effects of APC-N1 on cell-cell adheasion and E-cadherin. We found APC-N1 reduced cell-cell adhesion ability and expression of E-cadherin. Crystal violet staining、 QT-PCR and western blotting were used to detect the impacts of APC-N1 on cell-matrix adhesion and CD29. The results showed that APC-N1 increased cell-matrix adhesion and expression of CD29. P-catenin siRNA treatment led to elevated E-cadherin but had no effects on CD29 in MDCK-APC-N1 cells. PI3K inhibitor LY294002 treatment induced significant upregulation of E-cadherin along with reduced CD29 level. Taken together, APC-N1 influences MDCK cell adhesion through P13K/AKT signaling pathway.In conclusion, this study investigated the mechanism of APC-N1 on cilia formation and cell adhesion. The results showed that APC-N1 affected Hedgehog/Glil pathway by β-catenin and then upregulated AurA and HDAC6 levels, which resulted in cilia damage. Moreover, we found APC-N1 affected cell adhesion through PI3K/AKT signaling pathway.