The Study Of Hepatoma-Derived Growth Factor In Malignant Biological Behavior Of Human Hilar Cholangiocarcinoma

Background and significanceHilar cholangiocarcinoma is an uncommon but fatal tumor with poor prognosis, accounting for50%-70%of extrahepatic cholangiocarcinoma, and its morbidity has a worldwide increasing trend in recent30years. Surgical resection, either in the form of liver resection or liver transplantation, is the only effective curative therapy for hilar cholangiocarcinoma. However, few patients are candidates for curative resection because of the locally advanced nature of the tumor in adjacent organs and tissues, or insufficient remnant liver function. Recurrence rates are high even after curative resection. Adjuvant therapy (chemotherapy and radiation therapy) has not been clearly shown to reduce recurrence risk. So far the ideal tumor biomarkers with high sensitivity and specificity, used for early diagnosis and prognostic evaluation of hilar cholangiocarcinoma, have not been discovered or screened. Therefore, identifying the mechanism of its malignant biological behavior may help provide new therapeutic strategies.Hepatoma-derived growth factor (HDGF) is an acidic heparin-binding protein originally purified from cultured media with the human hepatoma cell line HuH-7. Several studies confirmed that HDGF has a variety of biological functions, promoting the proliferation and DNA synthesis of various cells, regulating the growth and development of liver, lung, kidney, heart, vessels and digestive tract, maintaining normal cell function, nourishing nerves and repairing tissues. Meanwhile, overexpression of HDGF was significantly associated with prognosis, tumor recurrence and some adverse clinicopathologic features. Previous studies proved that HDGF activates aggressive biological behaviors, including proliferation, invasiveness, and angiogenesis, as well as stimulates tumorigenesis by inducting the vascular endothelial growth factor (VEGF). Up to now, there is no report about HDGF expression in hilar cholangiocarcinoma, and so it has great significance to study the expression and malignant biological behavior of hilar cholangiocarcinoma.The extent of tumor angiogenesis is correlated with tumor aggressiveness and clinical outcome and is useful for predicting tumor recurrence. VEGF is the only growth factor most consistently found in a wide variety of conditions associated with angiogenesis. Several studies have shown that VEGF expression is closely related with MMP-9levels in breast cancer, non-small cell lung cancer, hepatoma, and gastric cancer, suggesting that the combined examination of VEGF and MMP-9in patients with gastric carcinoma offers promising prospects for predicting recurrence and prognosis. Gastric cancer ranks as the fourth most common malignancy and the second leading cause of cancer-related death worldwide with poor prognosis and high mortality. Since lymph node metastasis is an important prognostic factor in patients with gastric carcinoma, it is particularly important to exclude lymph node metastasis in the study of the development, recurrence, and survival of gastric cancer. Thus, our research group detected the expression levels of VEGF and MMP-9to find whether they could predict tumor recurrence and prognosis in pN0gastric cancer.Part one Expression And Clinical Significance Of Hepatoma-Derived Growth Factor In Human Hilar Cholangiocarcinoma Objective This study investigates the expression of HDGF combined with VEGF, their correlation with clinicopathologic features, and their prognosis in human hilar cholangiocarcinoma.Methods The expressions of HDGF and VEGF were analyzed by immunohistochemistry using the streptavidin peroxidase complex method for58patients with hilar cholangiocarcinoma receiving surgery. Their correlation with clinicopathologic features was then investigated. The relationships between them and the survival time of patients were retrospectively analyzed.Results HDGF and VEGF were positively expressed in27(46.6%) and42(72.4%) patients, respectively. HDGF and VEGF had a positive correlation (r=0.370, P=0.004) in the Spearman rank correlation analysis. HDGF expression was associated with gender and histological type. Patients with positive HDGF expression had a significantly poorer overall survival rate than those with negative HDGF expression (35.7%versus73.3%, P=0.003). Multivariate analysis showed that HDGF expression is an independent prognostic factor.Conclusion HDGF expression significantly correlates with VEGF expression and is a valuable prognostic factor for human hilar cholangiocarcinoma. Part two The In Vitro Study Of Hepatoma-Derived Growth Factor InMalignant Biological Behavior In Hilar Cholangiocarcinoma Cell Line FRH0201Objective To study the role of HDGF in the proliferation, migration, invasion and angiogenesis of human hilar cholangiocarcinoma cell line FRH0201in vitro.Methods After HDGF siRNA was transfected into FRH0201cells, Real time PCR and Western blot were used to detect the inhibition effect of siRNA on the expression of HDGF. Then, MTT assay was applied for detecting the survival ability of FRH0201cells; cell wound healing assay and migration assay were used for cell migration ability; Transwell-cabin assay was done for cell invasive ability; in vitro angiogenesis assay was used for the angiogenic ability of the FRH0201cells.Results48hours after the siRNA transfection, the results of PCR and Western blot showed that the expression of HDGF in FRH0201cells were inhibited both in mRNA and protein levels. MTT assay showed that the proliferation ability of FRH0201cells was only46.0%of the control cells(P<0.01); cell wound healing assay found that the migration ability was41.7%of the control cells(P<0.05); cell migration assay indicated that the migration ability was only26.5%of the control cells(P<0.01); transwell-cabin assay showed that the invasive ability of cells was only22.7%of the control cells(P<0.01); in vitro angiogenesis assay indicated that angiogenic ability of the FRH0201cells was only35.0%of the control cells(P<0.01).Conclusion HDGF plays an important role in promoting the malignant biological behavior (proliferation, migration, invasion and angiogenesis) of hilar cholangiocarcinoma cell FRH0201, and inhibition of HDGF expression could down-regulate the malignant biological behavior. Part three Correlation of Vascular Endothelial Growth Factor Expression With Tumor Recurrence and Poor Prognosis in Patients With pNO Gastric CancerObjective This study investigates the expression of VEGF combined with MMP-9, their correlation with clinical characteristics and their prognosis in patients with pNO gastric cancer after curative surgery. Methods A total of55patients enrolled in the study were analyzed by immunohistochemistry and their correlation with clinical characteristics was then investigated. Their relationships and the survival time of patients were retrospectively analyzed.Results VEGF and MMP-9were positively expressed in24(43.6%) and16(29.1%) patients, respectively, and had a positive correlation (r=0.324, P=0.016) in the Spearman rank correlation analysis. Univariate analysis showed that VEGF, MMP-9expression, vascular invasion, T stage and tumor size were associated with tumor recurrence, as well as disease-specific and overall survival rates. Patients with positive VEGF expression showed significantly higher recurrence, and poorer disease-specific and overall survival rates compared with those with negative VEGF expression. Multivariate analysis showed that VEGF expression, vascular invasion, T stage (serosal invasion), and tumor size were significant independent prognostic factors for tumor recurrence, disease-specific and overall survival in patients with pNO gastric cancer except of T stage for disease-specific survival.Conclusion VEGF expression, vascular invasion, T stage (serosal invasion), and tumor size can be used as valuable prognosticators in predicting tumor recurrence and prognosis for patients with pNO gastric cancer after curative surgery. Moreover, VEGF may have a synergistic effect with MMP-9during tumor angiogenesis, development, and progression.