整合素β4在肝细胞癌侵袭转移中的作用机制研究
发布时间：2019-05-09 06:17
【摘要】：目的：比较整合素β4(integrin β4, ITGB4)在人肝细胞癌(Hepatocellular carcinoma, HCC)组织及对应的癌旁组织中的表达差异,比较整合素β4在不同肝癌细胞系中的表达差异,分析整合素p4与肝癌临床病理学特征之间的联系,探讨整合素p4在肝癌侵袭转移中的作用。方法：选取华中科技大学同济医学院附属同济医院肝脏外科中心手术切除并经病理证实为肝细胞癌的手术标本68例,选取具有不同转移能力的肝癌细胞系7株,利用western-blot,免疫组织化学(immunohistochemistry, IHC)等方法检测整合素β4在癌与癌旁组织之间以及不同细胞系之间的表达情况,通过统计学方法分析整合素β4表达情况对肝癌临床病理特征的影响。结果：整合素p4在人肝癌组织中高表达,而在癌旁组织中低表达或不表达。癌组织中高表达的整合素β4与肿瘤的局部侵袭和低分化程度呈正相关。整合素β4在具有高侵袭转移能力的肝癌细胞系中(HLF, MHCC97L, MHCC97H, HCCLM3)高表达,而在低侵袭转移能力的肝癌细胞系中(Hep3B,HepG2,Huh7)低表达或不表达。结论：整合素β4在肝癌组织及具有高转移能力的肝癌细胞系中高表达,起促进肝癌侵袭转移的作用。目的：研究整合素β4(integrin β4, ITGB4)对肝癌细胞锚着非依赖性生长(anchorage independence)的影响以及调控该行为的机制。方法：选择高表达整合素β4的肝癌细胞系HCCLM3和HLF,用RNA干扰(RNAi)技术敲减整合素β4的表达,利用上述改造后的细胞系通过软琼脂克隆形成实验(soft agarose colony formation),失巢凋亡分析(anoikis assay)研究整合素β4对肝癌锚着非依赖性生长的影响。通过western blot,免疫共沉淀(Co-Immunoprecipitation, Co-IP),细胞免疫荧光等技术研究整合素β4调控的细胞信号通路对肝癌细胞锚着非依赖性生长的影响。结果：整合素β4增强肝癌细胞锚着非依赖性生长；整合素β4通过活化AKT/PKB信号通路来促进锚着非依赖性生长；整合素β4与表皮生长因子受体(epidermal growth factor receptor, EGFR)以配体非依赖的方式结合来促进锚着非依赖性生长；ITGB4-EGFR复合物通过FAK (focal adhesion kinase)而不是Src来激活AKT信号通路。结论：ITGB4-EGFR复合物通过活化FAK-AKT信号通路来促进肝癌细胞锚着非依赖性生长。目的：研究整合素β4在体内对肝癌生长及肺转移的影响。方法：利用裸鼠皮下成瘤模型研究整合素β4对肝癌在体内生长的影响；通过尾静脉注射肝癌细胞肺转移模型研究整合素p4对肝癌肺转移的影响。结果：ITGB4-EGFR活化的FAK-AKT通路可以促进肝癌在体内生长；ITGB4-EGFR活化的FAK-AKT通路促进肝癌肺转移。结论：ITGB4-EGFR-FAK-AKT信号通路促进肝癌体内生长及肺转移。
[Abstract]:Objective: to compare the expression of integrin 尾 4 (ITGB4) in human hepatocellular carcinoma (HCC) (Hepatocellular carcinoma, HCC) tissues and corresponding paracancerous tissues, and to compare the expression of integrin 尾 4 in different hepatocellular carcinoma cell lines. To analyze the relationship between integrin p4 and clinicopathological features of hepatocellular carcinoma (HCC) and to explore the role of integrin p4 in the invasion and metastasis of HCC. Methods: 68 cases of hepatocellular carcinoma were selected from the liver surgery center of Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. Seven hepatocellular carcinoma cell lines with different metastatic ability were selected and used western-blot,. Immunohistochemical (immunohistochemistry, IHC) and other methods were used to detect the expression of integrin 尾 4 between cancer and paracancerous tissues as well as between different cell lines. The effect of integrin 尾 4 expression on the clinicopathological characteristics of HCC was analyzed by statistical method. Results: integrin p4 was highly expressed in human hepatocellular carcinoma tissues, but low expression or no expression in paracancerous tissues. The high expression of integrin 尾 4 in cancer tissues was positively correlated with local invasion and low differentiation. Integrin 尾 4 was highly expressed in hepatocellular carcinoma cell line (HLF, MHCC97L, MHCC97H, HCCLM3) with high invasion and metastasis, but low expression or no expression in low invasion and metastasis hepatoma cell line (Hep3B,HepG2,Huh7). Conclusion: the high expression of integrin 尾 4 in HCC and HCC cell lines with high metastatic ability can promote the invasion and metastasis of HCC. Aim: to study the effect of integrin 尾 4 (ITGB4) on the Anchorage independent growth of (anchorage independence) in hepatocellular carcinoma cells and the mechanism of its regulation. Methods: hepatocellular carcinoma cell lines HCCLM3 and HLF, with high expression of integrin 尾 4 were selected to knock down the expression of integrin 尾 4 by RNA interference (RNAi) technique. The modified cell lines were used to form (soft agarose colony formation), by soft Agar cloning. The effect of integrin 尾 4 on Anchor-independent growth of Hepatocellular carcinoma was studied by (anoikis assay) analysis of out-of-nest apoptosis. Western blot, immunoprecipitation (Co-Immunoprecipitation, Co-IP) and cellular immunofluorescence were used to study the effects of integrin 尾 4-regulated cell signaling pathway on anchor-independent growth of HCC cells. Results: integrin 尾 4 enhanced anchor independent growth of HCC cells, and integrin 尾 4 promoted anchor independent growth through activation of AKT/PKB signaling pathway. Integrin 尾 4 binds to epidermal growth factor receptor (epidermal growth factor receptor, EGFR) in a ligand-independent manner to promote anchor-independent growth, and the ITGB4-EGFR complex activates the AKT signaling pathway through FAK (focal adhesion kinase) instead of Src. Conclusion: ITGB4-EGFR complex can promote the growth of HCC cells by activating FAK-AKT signaling pathway. Objective: to study the effect of integrin 尾 4 on the growth and lung metastasis of hepatocellular carcinoma (HCC) in vivo. Methods: the effect of integrin 尾 4 on the growth of hepatocellular carcinoma (HCC) in vivo and the effect of integrin p4 on the lung metastasis of HCC were studied by subcutaneously tumorigenic model of nude mice and the model of lung metastasis of HCC cells injected intravenously. Results: ITGB4-EGFR-activated FAK-AKT pathway could promote the growth of HCC in vivo, and ITGB4-EGFR-activated FAK-AKT pathway could promote lung metastasis of HCC. Conclusion: ITGB4-EGFR-FAK-AKT signaling pathway promotes the growth and lung metastasis of HCC in vivo.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2472541
[Abstract]:Objective: to compare the expression of integrin 尾 4 (ITGB4) in human hepatocellular carcinoma (HCC) (Hepatocellular carcinoma, HCC) tissues and corresponding paracancerous tissues, and to compare the expression of integrin 尾 4 in different hepatocellular carcinoma cell lines. To analyze the relationship between integrin p4 and clinicopathological features of hepatocellular carcinoma (HCC) and to explore the role of integrin p4 in the invasion and metastasis of HCC. Methods: 68 cases of hepatocellular carcinoma were selected from the liver surgery center of Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. Seven hepatocellular carcinoma cell lines with different metastatic ability were selected and used western-blot,. Immunohistochemical (immunohistochemistry, IHC) and other methods were used to detect the expression of integrin 尾 4 between cancer and paracancerous tissues as well as between different cell lines. The effect of integrin 尾 4 expression on the clinicopathological characteristics of HCC was analyzed by statistical method. Results: integrin p4 was highly expressed in human hepatocellular carcinoma tissues, but low expression or no expression in paracancerous tissues. The high expression of integrin 尾 4 in cancer tissues was positively correlated with local invasion and low differentiation. Integrin 尾 4 was highly expressed in hepatocellular carcinoma cell line (HLF, MHCC97L, MHCC97H, HCCLM3) with high invasion and metastasis, but low expression or no expression in low invasion and metastasis hepatoma cell line (Hep3B,HepG2,Huh7). Conclusion: the high expression of integrin 尾 4 in HCC and HCC cell lines with high metastatic ability can promote the invasion and metastasis of HCC. Aim: to study the effect of integrin 尾 4 (ITGB4) on the Anchorage independent growth of (anchorage independence) in hepatocellular carcinoma cells and the mechanism of its regulation. Methods: hepatocellular carcinoma cell lines HCCLM3 and HLF, with high expression of integrin 尾 4 were selected to knock down the expression of integrin 尾 4 by RNA interference (RNAi) technique. The modified cell lines were used to form (soft agarose colony formation), by soft Agar cloning. The effect of integrin 尾 4 on Anchor-independent growth of Hepatocellular carcinoma was studied by (anoikis assay) analysis of out-of-nest apoptosis. Western blot, immunoprecipitation (Co-Immunoprecipitation, Co-IP) and cellular immunofluorescence were used to study the effects of integrin 尾 4-regulated cell signaling pathway on anchor-independent growth of HCC cells. Results: integrin 尾 4 enhanced anchor independent growth of HCC cells, and integrin 尾 4 promoted anchor independent growth through activation of AKT/PKB signaling pathway. Integrin 尾 4 binds to epidermal growth factor receptor (epidermal growth factor receptor, EGFR) in a ligand-independent manner to promote anchor-independent growth, and the ITGB4-EGFR complex activates the AKT signaling pathway through FAK (focal adhesion kinase) instead of Src. Conclusion: ITGB4-EGFR complex can promote the growth of HCC cells by activating FAK-AKT signaling pathway. Objective: to study the effect of integrin 尾 4 on the growth and lung metastasis of hepatocellular carcinoma (HCC) in vivo. Methods: the effect of integrin 尾 4 on the growth of hepatocellular carcinoma (HCC) in vivo and the effect of integrin p4 on the lung metastasis of HCC were studied by subcutaneously tumorigenic model of nude mice and the model of lung metastasis of HCC cells injected intravenously. Results: ITGB4-EGFR-activated FAK-AKT pathway could promote the growth of HCC in vivo, and ITGB4-EGFR-activated FAK-AKT pathway could promote lung metastasis of HCC. Conclusion: ITGB4-EGFR-FAK-AKT signaling pathway promotes the growth and lung metastasis of HCC in vivo.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7
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