High Expression Of Secreted Protein Dickkopf-1and Its Role In Invasion And Metastasis In Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC), a primary adenocarcinoma arising from the epithelial cells of intrahepatic bile ducts, is the second commonest primary liver cancer (PLC). Its prognosis is more dismal than hepatocellular carcinoma (HCC) for the reason of higher incidence of recurrence and metastasis. It is important to explore the biomedical mechanism of recurrence and metastasis of ICC to improve its prognosis.Dickkopf-1(DKK1) is a secretory protein, which was firstly discovered in embryonic development. After that, DKK1had been regarded as a key factor in the carcinogenesis and cancer metastasis. It was also been found prognosis power among many malignant tumors. Meanwhile, elevating of serum DKK1has also been found as diagnostic marker in some cancers. The previous work of our institute had confirmed DKK1as a independent prognsis factor and accelerating invasion and metastasis in HCC. However, little is known about DKK1expression and its role in ICC.In this study, we explored the expression of DKK1in human ICC cell line and tumor tissues. The effects of knocking down DKK1with small interfering RNA (siRNA) in tumor progression and proliferation were also investigated. We further investigated the expression of DKK1and its clinical relevance and pronosis in138ICC patients with long-term follow-up by tissue microarrays (TMAs), in order to further the understanding of DKK1in ICC. Part1High expression of secreted protein Dickkopf-1in intrahepatic cholangiocarcinomaObjective:To investigate the mRNA and protein expression of DKK1in ICC tissues, serum samples and cell lines and its role in carcinogenesis of ICC.Methods:The expression of DKK1was evaluated in ICC tissues, preoperative serum samples and ICC cell line HCCC-9810their adjacent nontumorous tissues by qRT-PCR and western blot.Result:qRT-PCR and western blot results showed DKK1expression was elevated in ICC tissues (p<0.05). Early recurrence ICC had a high expression of DKK1(p<0.05). ELISA test showed that DKK1was also highly expressed in preoperative serum sample of ICC patients. In cell lines, DKK1expression in ICC cell line HCCC-9810was higher than that in intrahepatic biliary epithelial cells line IBEpiC.Conclusion:The secreted protein DKK1was elevated in ICC and may be related to the carcinogenesis of ICC.Part2Correlation and clinical significance of high expression of DKK1in intrahepatic cholangiocarcinomaObjective:To investigate the correlation and clinical significance of DKK1in cholangiocarcinoma.Methods:Immunohistochemistry was applied in the TMA of138ICC patients samples to detect the expression of DKK1, then the clinical significance of high DKKl expression was analyzed by SPSS19.Result:Overexpression DKK1in ICC was associated with hepatic hilar lymph nodes invasion(p<0.05). DKK1+patients had a worse OS and RFS than DKK1-patients. DKK1was an independent prognonsis factor in OS and RFS in multivariate analysis. Among early stage ICC patients, DKK1was also a prognostic factor. Combined serum CEA with DKK1can optimize the prognsis power.Conclusion:The high expression of DKK1is related with hepatic hilar lymph nodes invasion and could predict poor prognosis of ICC. Part3Relationship of DKK1with the invasion and metastasis of intrahepatic cholangiocarcinomaObjective:To investgate the relationship of DKK1with the invasion and metastasis of ICC cells.Method:small RNA interference was adopted to knock down the expression of DKK1in HCCC-9810. Immunofluorescence, qRT-PCR, western blot and ELISA were used to confirm the knock down the expression of DKK1. The ability of proliferation, invasion and migration of ICC cells was tested by CCK8, transwell and wound healing test after knock down of DKK1.Result:DKK1expression was successfully down regulated by siRNA. The proliferation of ICC cells was not significantly influenced by the down regulation of DKK1(p>0.05). Low DKK1expression ICC cells was accompanied by impairment in invasiveness and migration (p<0.05).Conclusion:The secreted protein DKK1was positive correlation with the ability of invasion and metastasis of ICC.Part4High expression of DKK1promotes lymphatic metastasis via VEGF-C and MMP9in ICCObjective:To expatiate the mechanism of DKK1in promoting lymphatic metastasis via VEGF-C and MMP9in ICC.Methods:We investigated the corelationship of mRNA expression of DKK1, VEGF-C and MMP9in13ICC tissues. Immunohistochemistry of DKK1, VEGF-C and MMP9was performed and analysed in TMAs containing138ICC samples. Changing mRNA levels of VEGF-C and MMP9were also evalutaed in siRNA DKK1downregulated ICC cell line HCCC-9810.Result:In fresh tissues and TMA, DKK1expression was positively correlated with VEGF-C and MMP9(p<0.05). After down regulation of DKK1by siRNA in ICC cells, VEGF-C and MMP9expression were also decreased (p<0.05).Conclusion:DKK1may promote lymphatic metastasis via VEGF-C and MMP9in intrahepatic cholangiocarcinoma.