Effects Of LI-cadherin On Biological Function And Wnt/β-catenin Pathway Of Colon Cancer LoVo Cells

Background:Colon cancer is one of the most common malignant tumors in China and fourthly ranks in leading causes of cancer-related death.Despite advanced treatment regimens,such as surgery,chemotherapy,immunotherapy,can improve the survival of colon cancer patients,tumor recurrence or distant metastasis still occurs,and the overall prognosis of patients is still unsatisfactory.Therefore,exploring the mechanism of invasion and metastasis of colon cancer is of great clinical significance and practical value for cancer precaution and treatment.Liver-Intestine cadherin(LI-cadherin,cadherin-17)is a unique structural member of the cadherin family.It has calcium(Ca2+)dependent cell adhesion function,independent of cytoskeleton interaction.Studies have found that the abnormal expression of LI-cadherin in colon cancer is related to cell invasion and metastasis,but the specific molecular mechanism is not clear.Early experiments have found that LIcadherin could regulate all three Wnt signaling pathway-related genes in colon cancer cells.Based on this finding,this study will focus on canonical Wnt/β-catenin pathway,aiming for exploring the effect of LI-cadherin upon Wnt/β-catenin pathway,and the correlation with biological behavior of colon cancer regulated by LI-cadherin.Objective:Exploring the specific regulation mechanism of LI-cadherin on Wnt/β-catenin signaling pathway.To clarify the correlation between the regulatory effect of LIcadherin on Wnt/β-catenin signaling pathway and its biological behaviour of colon cancer.Method:1.The lentivirus silencing vector and adenovirus overexpression vector were constructed,and the vectors were packaged and infected to LoVo cells respectively.Hence,cell models for LI-cadherin up-regulation and down-regulation were established.2.The MTS and Transwell assays were performed to detect the effect of LIcadherin in proliferation,invasion and migration of colon cancer cells.3.Western Blot was used to detect the expression levels of Wnt/β-catenin signaling pathway,and the expression of β-catenin in nucleus and cytoplasm.4.Dual luciferase reporter assay was conducted to detect the activity of TCF/LEF promoter in LoVo cells.5.Co-Immunoprecipitation(Co-IP)analysis was used to confirm the protein that interacts with LI-cadherin in LoVo cells.Results:1.Overexpression of LI-cadherin could inhibit the proliferation,migration and invasion abilities of colon cancer cells(P<0.01).While LI-cadherin knockdown cells exhibited opposite effects(P<0.01).2.Overexpression of LI-cadherin in LoVo cells could increase the expression level of numerous proteins,such as FZD3、APC、GSK3β、β-catenin、p-β-catenin、MYC、Cyclin D1、PPARD(P<0.05).Besides,the upregulated expression of LI-cadherin in LoVo cells reduced the expression level of p-GSK3β(P<0.01)and induced the protein level of β-catenin in nucleus(P<0.01).While LI-cadherin knockdown cells showed different results.3.Down-expression of LI-cadherin in LoVo cells could reduce the activity of TCF/LEF promoter(P<0.001).However,there was no significant change in LIcadherin overexpressed cells(P>0.05).4.β-catenin and GSK3β were detected in the protein complex from the immunoprecipitation by using anti-LI-cadherin antibody.Their expressions were directly proportional to LI-cadherin.5.The canonical Wnt signaling pathway inhibitor XAV939 could rescue the upregulated expression of total β-catenin and nuclear content in LI-cadherin overexpression cells(P<0.05).The canonical Wnt signaling pathway agonist LiCl could reverse the downregulated expression of total β-catenin and nuclear content in LI-cadherin knockdown cells(P<0.01).6.Overexpression of LI-cadherin exerted inhibitory effect on the biological function of LoVo cells,which could be rescued by LiCl.While XAV939 could reverse the malignant behavior of cells caused by downregulated LI-cadherin.Conclusion:1.LI-cadherin inhibited the proliferation,invasion and metastasis of colon cancer.2.LI-cadherin could activate Wnt/β-catenin signaling pathway.3.The effect of LI-cadherin upon the Wnt/β-catenin signaling pathway did not play a decisive role in the occurrence and development of colon cancer.