UBC9 Affects Invasion And Metastasis Of Colorectal Cancer Cells Through β-catenin Signalling

Objective:Colorectal cancer cells were used as the research object to detect the expression of UBC9,and to investigate the effect of UBC9 on invasion and metastasis of Colorectal cancer cells.By transfecting UBC9 shRNA interference vector and UBC9 overexpression vector,the molecular mechanism of UBC9 on Colorectal cancer cell invasion and migration was studied,which provided a new therapeutic target for the treatment of Colorectal cancer.MethodsThe expression levels of UBC9 protein and mRNA in Colorectal cancer cell lines: SW480,HCT116,DLD-1,LOVO and Colorectal canceric epithelial cell NCM460 were detected by Western Blot and Real time QPCR.UBC9-expressing higher Colorectal cancer cells were transfected with UBC9-shRNA,and UBC9 protein and mRNA expression levels were detected after transfection;cell invasion and migration assays and cell wound healing assays were used to detect cell migration and invasion;Western Blot detection of MMP2 Changes in the expression of MMP9 protein.Then UBC9-shRNA and UBC9 overexpression vectors were transfected into Colorectal cancer cells,Western Blot detecting the expression changes of EMT-related proteins Vimentin、Snail1、E-cadherin and the Wnt/β-catenin signalling proteins TCF4、β-catenin,、GSK3β、p-GSK3β.Results1.The protein and mRNA expression level of UBC9 in human Colorectal carcinoma cell lines SW480,HCT116,DLD-1 and LOVO was significantly higher than that in human normal Colon epithelial cells NCM460(P<0.01),while the expression of SW480 and HC116 was the highest.We chose these two cells for the following experiments.2.Transfection of UBC9 shRNA interference vector interfered with UBC9 expression;we found that shUBC9 a could effectively interfere with the expression of UBC9 mRNA and protein(P<0.01).3.Compared with the empty vector group,wound healing assay and cell migration and invasion assay showed that down-regulation of UBC9 expression can inhibit the healing speed,migration and invasion of SW480 and HCT116(P<0.01),and the expression of MMP2 and MMP9 protein was inhibited(P<0.01).There was no difference between the empty vector group and the normal group(P>0.05)。4.Down-regulating UBC9 inhibited the protein expression of Snail and Vimentin in SW480 and HCT116 cells,and the expression of E-cadherin was up-regulated(P<0.01).Up-regulation of UBC9 promoted the expression of Snail and Vimentin in SW480 and HCT116 cells,and the expression of E-cadherin expression was inhibited(P<0.01).There was no difference between the normal group and the empty vector group(p>0.05).5.Down-regulating UBC9 inhibited the protein expression of β-catenin and TCF4 in SW480 and HCT116 cells,and up-regulated the expression of p-GSK3β(P<0.01).Up-regulation of UBC9 promoted β-catenin and TCF4 expressions,and the expression of p-GSK3β was inhibited(P<0.01).There was no change in the expression of GSK3β in each group(p>0.05).There was no difference between normal group and empty vector group(p>0.05).ConclusionInhibiting the UBC9 expression of Colorectal cancer cells,inhibits their invasion and migration,inhibits epithelial-mesenchymal transition,and inhibits the Wnt signaling pathway.UBC9 may promote invasion,migration and epithelialmesenchymal transition of Colorectal cancer cells through the Wnt/β-catenin pathway.