Abnormal Expression And Function Of Cx43 Of Tumor Cells In The Interaction Between CRC Cells And Stromal Fibroblasts

Colorectal cancer(CRC)is the most frequently diagnosed cancer worldwide.Tumor microenvironment plays an important role in eancer progression.Cancer associated fibroblasts(CAFs)are the most common components in the tumor microenvironment.In our previous study,we found that CAFs promoted the invasion and migration of CRC cells,while normal fibroblasts(NFs)inhibited them.Connexin 43(Cx43)might be involved in the process.In order to explore the function and the potential mechanism of Cx43 in the interaction of tumor cells and fibroblasts,we continued to establish the crosstalk model between CRC cells and fibroblasts.CRC cell lines SW620,SW480,RKO and human embryo lung fibroblast HELF,HELF activated by TGF-β as well as human primary fibroblasts were choosed to perform the experiment.E-cadherin expression was up-regulated in SW620 cells which were co-cultured with supernatant of NFs for 4d by Western Blotting.In the same conditions,Fibronectin and Vimentin were down-regulated,especially Vimentin.Meanwhile,the expression of Phospho-β-catenin(s552)and DVL3 were down-regulated,especially s552.Wnt signaling pathway was inhibited when CRC cells were co-cultured with supernatant of NFs for 2d by Dual luciferase reporter assay.The expression of Cx43 in tumor cells was significantly up-regulated in SW620 cells which were co-cultured with supernatant of fibroblasts for 4d by Western Blotting,particularly with CAFs for 4d.Experimentally,it was verified that the expression of Cx43 was knocked down in SW480 cells and overexpressed in SW620 and RKO cells by Western Blotting.The migration and invasion of SW480 cells with low Cx43 expression in cytoplasm enhanced,while the migration and invasion of SW620 cells with high Cx43 expression in cytoplasm weakened by Transwell assay.The proliferation of RKO cells with high expression of Cx43 in cytoplasm weakened by CCK8 asssy.Gene Set Enrichment Analysis(GSEA)indicated that the low expression of Cx43 in cytoplasm was associated with Wnt signaling pathway in RKO cells.CRC patients with high expression of GJA1(encoding Cx43)had a poor prognosis in the TCGA and GEO database.Correlation analysis revealed that there was a negative correlation between GJA1 and CDH1(encoding E-cadherin).GJA1 was positively correlated with CDH2(encoding N-cadherin)and VIM(encoding Vimentin).TCGA database information confirmed that GJA1 is positively correlated with CTNNB1(encoding β-catenin).Co-immunoprecipitation studies clearly showed that Cx43 could interact with p-catenin.Though the expression of Cx43 was only up-regulated in cytoplasm in SW480 and SW620 cells which co-cultured with supermatant of NFs for 4d,supernatant of CAFs could promote Cx43 co-located withβ-catenin into the nucleus by Confocal laser scaning microscopy.TGF-β promoted Cx43 into the nucleus by Western Blotting.The migration and invasion of CRC cells with nuclear expression of Cx43 were enhanced by Transwell assay.Dual luciferase reporter assay confirmed that CAFs promoted the activation of Wnt signaling pathway in SW620 cells.TGF-β showed the same results.Through the above experiments,we can get the following conclusions:1.NFs inhibit CRC cells EMT and Wnt signaling pathway,while CAFs promote the activation of Wnt signaling pathway.2.The interaction of fibroblasts and colorectal cancer cells significantly increase the expression of Cx43 in tumor cells.3.The migration,invasion,proliferation and Wnt signaling pathway of colorectal cancer cells with high expression of Cx43 in cytoplasm are inhibited,but that with nuclear expression are increased.4.CAFs promote Cx43 into the nuclei of tumor cells by TGF-β,where Cx43 interacts with β-catenin.