| Objective Cell proliferation and tumor metastasis are considered as the main reasons for death in colorectal carcinoma(CRC).IRE1α-XBP1 pathway is the most conserved UPR pathways,which are activated during ER stress caused by the accumulation of unfolded or misfolded protein in the lumen of ER.Here,we demonstrated the critical role of IREla-XBP1 pathway and underlying molecular mechanism in cell proliferation and tumor metastasis in CRC.Method In order to investigate the expression status of ERE1α in CRC,immunohistochemical staining for IRE la was conducted on a CRC TMA containing 49 pairs of CRC specimens and corresponding normal colorectal mucosal tissues.Furthermore,the prognostic value of IRE1α was analyzed in TMA.Lentivirus-mediated shRNA to knockdown IRE1α and IRE1α overexpressed modles in HCT116 and SW620 cells were constructed.Real-time qPCR and Western blot analyses were conducted to detect the expression level of IRE1α,XBP1 and Cyclin Dl.CCK8 assay was applied to detect the effect of IRE la on cell proliferation.Matrigel invasion assays were applied to detect the effect of IRE1α on the invasion ability of CRC cells.ChIP experiments were conducted to identify a XBP1s-binding site in Cyclin D1 promoter.Immunofluorescence staining was conducted to determine the expression levels of epithelial marker E-cadherin and mesenchymal marker N-cadherin in HCT116 cells.Results By the use of tissue microarray analysis of samples from 49 patients with CRC,IRE la was determined to be an independent predictor of overall survival as higher expression of IRE la in CRC patients showed lower survival rates(p=0.0041).RNA interference and ectopic expression of IRE1α were applied to determine the molecular effects of IRE la in CRC cells.The silencing of IRE la inhibited the proliferation and blocked the invasion of CRC cells in vitro,while ectopic expression of IREla in turn promoted cell proliferation and invasion.IREla-XBP1 pathway regulated the mitosis of CRC cells through the directly binding of XBP1s to Cyclin D1 promoter to activate Cyclin D1 expression.With the silencing of IREla,E-cad increased dramatically with the decrease of N-cad,while the trends of the two proteins were reversed in IREla overexpressed cells,which indicated that the promotion effects of IRE1α-XBP1 pathway in metastasis of CRC were through inducing EMT.Conclusion Our results reveal that IRE1α-XBP1 pathway plays an important role in tumor progression and epithelial-to-mesenchymal transition(EMT),and IRE1α could be employed as a novel prognostic marker and a promising therapeutic target for CRC. |
