Function And Mechanism Of XBP1s In The Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common liver cancer,the third leading cause of cancer-related death and the sixth most common cancer worldwide.It is estimated that each year,half a million new cases are diagnosed worldwide.It was reported that developing countries have the highest prevalence of the disease.In early stage HCC,potential curative treatments including surgical resection,percutaneous ablation andliver transplantation are available.However,for advanced HCC,curative treatments are not available and the 5-year survival for advanced stage patients is poor.Accumulating evidence has demonstrated that abnormal expression and mutation of genes are involved in the carcinogenesis and progression of HCC,including cyclin D1(CCND1),epidermal growth factor receptor(EGFR),c-myc and Ras,as well as mutations of tumor-suppressor genes.Recently,several proteins and signaling pathways have been found to be correlated with the prognosis of HCC.Tumor metastasis and recurrence are the primary contributors to poor prognosis in patients with hepatocellular carcinoma(HCC).The mechanisms of metastasis and recurrence in HCC patients have not been thoroughly understood,so explorations of prognostic molecular markers for this malignancy are still essential.The epithelial-mesenchymal transition(EMT)of tumor cells is the predominant mechanism of HCC progression.XBP1 s is a newly discovered molecule involved in the endoplasmic reticulum(ER)stress response,which is an adaptive response and defense mechanism in cells that enables survival under adverse conditions.Twist is a member of the original helix-loop-helix(bHLH)transcription factor family.It is one of the key inducers of EMT,a transdifferentiation procedure associated with embryonic development and tumor metastasis.It plays a pivotal role in the regulation of EMT.Mechanistically,the binding of Twist to E-box can inhibit the expression of E-cadherin by transcription,thereby destroying the intercellular adhesion and inducing the spread of individual cancer cells from the primary site,further leading to the activation of mesenchymal markers and Subsequent induced cell invasion.Abnormally high XBP1 s expression has been found in tumor cells,but the role of XBP1 s in HCC progression remains unclear.We found that the expression of XBP1 s in HCC cell lines and tissue samples was higher than that in control cells and tissue samples.Clinicopathological analysis showed that the expression of XBP1 s was closely correlatedwith distant metastasis and poor prognosis in HCC.In vivo and in vitro experiments confirmed that the overexpression of XBP1 s promoted EMT and metastasis in HCC cells.XBP1 s silencing attenuated cellular migration and development of the EMT phenotypein vitro.Through further study to elucidate the molecular mechanism underlying the promotion of EMT by XBP1 s in HCC cells,we confirmed that XBP1 s could mediate the expression of Twist.In HCC cells,XBP1 s enhanced the expression of Twist and Snail,resulting in a subsequent reduction in the expression of E-cadherin,a contributor to cell-cell adhesion.Overall,this study reveals a novel XBP1 s / Twist / Snail axis that mediates EMT in HCC cells and the invasion and metastasis of HCC.Part 1 The expression of XBP1 s in all four HCC cell lines and normal liver cellsObjective: To study the difference of XBP1 s expression in all four HCC cell lines and normal liver cells.To study the expression of XBP1 s in clinical HCC tissues and the relationship of XBP1 s expression and survival of patients with HCC.Methods: 1.By using Western blot analysis,IF analysis and qRT-PCR,we detected the XBP1 s expression in various human HCC cells lines.2.By using Western blot analysis,immunohistochemistry and qRT-PCR,we detected the expression of XBP1 s in HCC and the surrounding tissues.Results: 1.The expression of XBP1 s protein was increased in all four kinds of human HCC cells lines compare with the QZG cell line(p < 0.01).Immunoflourence analysis revealed that XBP1 s significantly increased in HepG2 cells compare with the QZG cells,and the staining patterns of XBP1 s was predominantly located in the nuclei of hepatocytes.2.The protein of XBP1 s expression was significantly increased in HCC tissues confirmed by western blot assay(p < 0.01).Immunohistochemistry showed that XBP1 s was mainly located in the nucleus of liver cells.The positive expression of XBP1 s in HCC tissues at a value(76.7%,79 of 103)was significantly higher than that in adjacent cirrhosis tissues(19.4%,20 of 103)(p < 0.05).Overexpression of XBP1 s was closely correlated with tumor size(p = 0.028),intrahepatic invasion(p = 0.003),and distance metastasis(p = 0.039).However,there is no significant correlation with gender,age,?-fetoprotein and HBV infection.Conclusions:The expression of XBP1 s in HCC cell lines and tissue samples is associated with poor prognosis.Part 2 XBP1 s enhances invasive and metastatic potential of HCCObjective: To evaluate whether XBP1 s was responsible for the greater migratory and invasive capabilities of XBP1s-SMMC7721 and XBP1s-HepG2 cells.Methods: 1.By using Matrigel invasion assays,we detected the invasiveness of the XBP1s-treated cells.2.By using scratch wound healing,the cell migration rate between HepG2 / XBP1 s and HepG2 / GFP cells was compared.3.We examined the metastasis assay in nude mice to verify the effect of XBP1 s on tumor metastasis in vivo by injecting the XBP1s-HepG2 cells into nude mice via tail vein.Results: 1.The Matrigel invasion assay showed that the invasiveness was markedly increased in the XBP1s-treated cells compared with the empty vector-transfected cells(p < 0.05).2.Compared with the empty vector controls,overexpression of XBP1 s increased the cell motility in HepG2 cells(p < 0.05).3.More micro-metastatic lesions were detected in the lungs of nude mice injected with XBP1s-HepG2 compared with the control group(p < 0.05).Conclusions: XBP1 s enhances invasive and metastatic potential of HCC.Part 3 XBP1 s promotes EMT in HCC cellsObjective: To study the role of XBP1 s in regulating EMT in HCC cells.Methods: 1.We analyzed the expression of XBP1 s,E-cadherin,?-catenin and Vimentin by western blot in HepG2 cells transfered XBP1 s and pcDNA-control.2.By using immunofluorescence staining,we detected the relationship between the expression of XBP1 s and the expression of epithelial markers(E-cadherin and ?-catenin)and mesenchymal marker(snail)in HepG2 cells.3.We analyzed the expression of XBP1 s,E-cadherin and Vimentin by western blot in HepG2 cells transfered XBP1 s and pcDNA-control in clinical HCC tissues.Results: 1.Up-regulation of XBP1 s decreased expression of epithelial markers(E-cadherin and ?-catenin)and increased expression of mesenchymal marker(Vimentin)in HepG2 cells(p <0.05).2.Up-regulation of XBP1 s decreased expression of epithelial markers(E-cadherin and ?-catenin)and increased expression of mesenchymal marker(snail)in HepG2 cells by immunofluorescence staining(p <0.05).3.The expression of XBP1 s was positively correlated with Vimentin(p < 0.001)and is negatively correlated with E-cadherin expression by linear analysis(p < 0.001).The relationship between XBP1 s expression and epidermal-mesenchymal marker was further analyzed.Among the HCC samples with high expression of XBP1 s,the high expression of Vimentin and the lowexpression of E-cadherin were 66.7% and 64.5.Among the HCC samples with low expression of XBP1 s,the high expression of Vimentin and the low expression of E-cadherin were only 11.1% and 18.2%.There was a significant difference between the two groups(p < 0.05).Conclusions: XBP1 s promotes EMT in HCC cells.Part 4 XBP1 s regulates EMT in HCC cells via Twist / Snail pathwayObjective: To examine the mechanism of Twist expression were influenced by XBP1 s.Methods: 1.By using immunofluorescence staining,we investigated whether XBP1 s up-regulated Twist expression in HepG2 cells.2.Western blotting was used to further determine whether XBP1 s induced Twist and its downstream gene expression.3.By using Twist promoter luciferase reporter assay,we analyze the XBP1s-induced Twist transcriptional activation.4.Western blotting was used to further investigate whether XBP1 s participate in EMT of hepatocellular carcinoma cells through the Twist/Snail pathway.Results: 1.The expression of Twist was significantly increased in XBP1s-overexpressing cells by immunofluorescence staining.2.The experimental data in this study showed that the expression of both Twist and Snail was significantly increased and the expression of E-cadherin was significantly decreased in XBP1s-treated HepG2 cells.Meanwhile,the expression of both Twist and Snail was significantly decreased and the expression of E-cadherin was significantly increased in HepG2 cells transfected with XBP1 s siRNA.3.The luciferase reporter assay showed that the luciferase activity in HepG2 cells transfected with the(-14 to-20)mutant vector was significantly reduced compared to the Twist wild-type promoter vector(p < 0.01).4.Western blotting results showed that the expression of Twist was effectively inhibited in cells transfected with Twist siRNA,nd partially reversed the EBP phenotype induced by XBP1 s.The expression of Snail was effectively inhibited in cells transfected with Snail siRNA,and partially reversed the EBP phenotype induced by XBP1 s.Conclusions: XBP1 s could induce EMT to play an important role in HCC metastasis by the XBP1 s / Twist / Snail-dependent pathway.XBP1 s can be served as a novel regulator of EMT and cell invasion.