The Effect Of Bmi1 Expression On Proliferation And Differentiation Of Hepatic Progenitor Cells And Angiogenesis Of Liver Tumors

Background and Aims:Hepatocellular carcinoma(HCC)is a global problem and the second most common cause of cancer related deaths in the world.Its global incidence has been reported to be on the rise and is predicted to exceed a million cases per year by 2025.The overall survival of patients with HCC is dismal with a five-year survival of less than 15%.This is mainly due to liver cancer showed the tumor heterogeneity.Angiogenesis is a necessary condition for tumor formation and metastasis,It is found that microvessel density is related to tumor growth and metastasis and has a significant correlation with the prognosis of various human tumors.Hepatocellular carcinoma is a highly abundant blood-rich tumor,Cell carcinogenesis,development,metastasis play an important role.We have found that the activation of Hepatic progenitor cells(HPCs)is significantly associated with the recurrence and prognosis of postoperative Combined Hepatocellular-Cholangiocarcinoma(CHC),and the proliferation status of Ductular reaction(DR)(Proliferative index of Ductular reaction,PIDR)has a significant positive correlation with the activation of hepatic progenitor cells(Hepatology,2012),suggesting that the stemness of the adjacent of cancer can affect the heterogeneity of the tumor.Bmi1 may be involved in cell proliferation and differentiation,regulation of apoptosis,and the development of a variety of tumors are closely related.Therefore,the purpose of this study is to clear: the expression and activation of hepatic Bmi1 precursor cells on the prognosis of patients with hepatocellular carcinoma and tumor angiogenesis is associated with liver Bmi1 expression;whether the progenitor cell activation and differentiation;What is the mechanism by which the cancer is affected by the side tissue of the cancer,and whether it is the mechanism by which angiogenesis is involved.Methods:The first part retrospectively collected the clinical prognosis and pathological data of 70 patients who were diagnosed as Combined Hepatocellular-Cholangiocarcinoma(CHC)and underwent surgical treatment from October 1996 to May 2008 in the Hepatobiliary Surgery Hospital of the Second Military Medical University.Surgical pathology specimens were provided by the Institute of Pathology.The levels of microvessel density(MVD),Bmi1 expression in non-tumor tissue were observed by immunohistochemistry using anti-CD34,Bmi1,Ep CAM,CK7 and PNCA antibodies.The levels of MVD,Bmi1,Ep CAM,K7-DR and PIDR expression were analyzed by single factor and multivariate analysis.Finally,the correlation between these histological indexes on MVD expression and analysis was analyzed.Expression of the correlation between the analysis.In view of the results of the first part,the effects of Bmi1 on the specific activation and differentiation of Hepatic progenitor cells were examined in vivo and at the cells.The mechanism of Bmi1 on cancer angiogenesis was also explored.The Bmi1 overexpressed cell lines of Hepatic progenitor cell m HPC and BNLCL2 were constructed by lentiviral transfection technique.The changes of proliferation and metastasis of cell lines were observed and the possible differentiation directions were observed.The proliferation activity of the cell line was detected by CCK-8assay.The colony forming ability was detected by clonal formation assay.Transwell assay was used to detect the cell metastasis ability.The expression of CD133 and SOX9,CK7 of bile duct markers,and the expression of fibrosis index ?-SMA were detected by Real-time PCR and Western-Blot and immunofluorescence.The overexpressing Bmi1 Hepatic progenitor cells line and mouse hepatocarcinoma cells were combined in nude mice subcutaneously and compared with mice injected with mouse hepatocellular carcinoma alone.After 3 weeks,the tumor was taken and the MVD level was evaluated by immunohistochemistry.Real-time PCR Screening of possible angiogenesis-related factors and validation by Western-Blot.Bmi1+/-and Bmi1+/+ control mice were used to inoculate the hepatoma of mice in the liver subcutaneously in nude mice to establish the implanted tumor model to explore the effect of Bmi1 on tumor-related biological characteristics and angiogenesis.Results:1.The number of tumors>1 and the size of tumors>5cm were the risk factors for the overall survival of patients with Combined Hepatocellular-Cholangiocarcinoma.Intraoperative hemorrhage >300ml,high TNM stage,high expression of PIDR more than 50%,non-tumor Bmi(3+),MVD>60/mm2 are independent risk factors affecting the overall survival of patients after operation.2.MVD in patients with Combined Hepatocellular-Cholangiocarcinoma(CHC)was associated with the expression of Bmi1 and PIDR.3.Overexpression of Bm1 can promote the proliferation and metastasis of Hepatic progenitor cells.4.Bmi1 can promote the activation of hepatic precursor cells and promote the expression of CK7,fibrosis index ?-SMA and CD133 and SOX9 of stemness index.5.Bmi1 can promote the expression of b FGF,VEGFA and VEGFC in the angiogenesis of Hepatic progenitor cells.Conclusions:Statistical analysis of clinical data showed that MVD,Bm1 and PIDR were significantly correlated with the prognosis of patients with mixed hepatocellular carcinoma,and the correlation between MVD and the expression of Bmi1 and PIDR in patients with liver cancer.Further verification by in vivo and in vitro experiments found that Bmi1 expressed high levels of progenitor cells in the liver may be through the secretion of angiogenesis related factors affect tumor angiogenesis;Bmi1 promotes the expression of hepatic progenitor cells into bile duct fibroblasts and related markers.It is suggested that the expression of Bmi1 may promote the development of tumor through the activation of HPCs.