ARK5在肝癌组织中的表达及对肝癌细胞增殖、凋亡和侵袭的影响

发布时间：2018-01-31 13:43

本文关键词： 肝细胞癌 ARK5 TGF-β1 Akt　出处：《郑州大学》2016年博士论文　论文类型：学位论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)为世界范围内最常见的恶性肿瘤之一,有研究报道HBV和HCV感染为HCC最主要的病因,在欧美等其他一些发达国家或地区,酒精性肝硬化以及与肥胖相关的非酒精性脂肪肝亦是造成罹患HCC的主要原因。恶性肿瘤的发生和发展通常是由体内外多种因素共同作用的多阶段参与的复杂过程。HCC的发生过程是由体内外多种因素以及多条信号通路共同作用的结果,尽管目前临床已广泛使用常规放疗以及化疗等技术有效控制了HCC的发生和发展,但大部分患者在接受上述疗法之后的预后情况依然不容乐观。靶向治疗是目前新发现的、能够有效治疗HCC的新途径,已成为基础肿瘤医学界研究的热点和难点内容。广大的医学工作者们正在试图寻找能够成为临床上有效治疗HCC的新靶点,以期为有效控制HCC和改善患者预后奠定基础。ARK5(AMPK-related protein kinase 5)的异常表达与临床上多种恶性肿瘤的发生和发展存在密切联系,还与恶性肿瘤的侵袭和转移相关。目前研究已证实ARK5基因在人类的多种恶性肿瘤组织中存在高表达,特别是在一些侵袭和转移性较高的恶性肿瘤中,但其在HCC中表达研究相对较少。目前对于Akt(Protein Kinase B)在HCC发生和发展过程中如何调控ARK5与HCC肿瘤的增殖、凋亡以及侵袭过程中的相关机制的研究依然较少,TGF-β1(transforming growth factor-beta 1)信号通路出现异常是恶性肿瘤发生、发展、浸润、转移等过程的重要因素,TGF-β1信号通路出现异常是恶性肿瘤发生、发展、浸润、转移等过程的重要因素。本研究拟观察ark5在hcc患者肿瘤组织中的表达及意义,分析ark5表达的变化对hcc肿瘤细胞增殖、凋亡、侵袭的影响,探究tgf-β1和akt信号活化对hcc肿瘤细胞中ark5表达的调控作用及其对hcc肿瘤细胞生物学行为的影响,旨在为了解hcc的发生和发展机制并为hcc的临床诊断及靶向治疗提供可靠依据。目的本研究拟观察ark5在肝细胞癌(hepatocellularcarcinoma,hcc)患者肿瘤组织中的表达及意义,分析ark5表达的变化对hcc肿瘤细胞增殖、凋亡、侵袭的影响,探究tgf-β1和akt信号活化对hcc肿瘤细胞中ark5表达的调控作用及其对hcc肿瘤细胞生物学行为的影响,旨在为了解hcc的发生和发展机制并为hcc的临床诊断及靶向治疗提供可靠依据。方法(1)运用荧光定量pcr对20例hcc组织以及相应癌旁正常组织样品中ark5mrna的表达情况进行测定;运用westernblot法检测hcc癌组织及其相应癌旁正常组织中ark5蛋白的表达;运用免疫组织化学法检测样品中ark5蛋白的表达;分析ark5蛋白的表达与hcc患者临床病理参数以及患者生存率的相关性。(2)将sirna-ark5转染人肝癌细胞ammc-7721,以此降低细胞中ark5的表达;荧光定量pcr和westernblot法检测转染后ammc-7721细胞中ark5mrna和相应蛋白的表达;运用mtt法检测转染后细胞的增殖活性;annexinv-fitc/pi双染法检测转染对细胞凋亡的影响;用transweii小室法检测转染后细胞的侵袭能力;细胞划痕实验检测转染后对细胞迁移能力的影响;检测转染后细胞中caspase-3活性。(3)将tgf-β1抑制剂ly364947作用于人肝癌细胞ammc-7721,以此降低细胞中tgf-β1的表达,brdu细胞增殖实验检测细胞的增殖能力;用transweii小室法检测细胞的侵袭能力;细胞划痕实验检测细胞的迁移能力;westernblot法检测细胞中tgf-β1、akt、ark5蛋白的表达。结果(1)ARK5 mRNA及其相应蛋白在HCC癌组织中的表达显著高于其在相应癌旁正常肝脏组织中的表达;ARK5高表达与HCC肿瘤大小、组织分化程度、肿瘤分期显著相关(P0.05);而与患者年龄、肝硬化、HBsAg、血清AFP无关(P0.05);ARK5蛋白表达能够作为HCC的一个独立预后判断因素。(2)转染siRNA-ARK5后AMMC-7721细胞中ARK5 mRNA和相应蛋白的表达显著降低(P0.05);siRNA-ARK5组AMMC-7721细胞的增殖活性显著低于siRNA-NC组和空白组,且siRNA-ARK5组细胞生长抑制率在48 h为最高,与siRNA-NC组相比差异显著(P0.01);转染48 h后,siRNA-ARK5组细胞的凋亡率明显增高,与空白组相比差异显著(P0.01);siRNA-ARK5组细胞的侵袭能力明显降低,与与空白组相比差异显著(P0.05);与空白组相比,siRNA-ARK5组细胞的迁移能力明显降低(P0.01);siRNA-ARK5组细胞的caspase-3活性较空白组明显增高(P0.05)(3)TGF-β1抑制剂Ly364947作用于AMMC-7721细胞后,TGF-β1 inhibitor组细胞的增殖活性较对照组明显降低(P0.01);TGF-β1 inhibitor组细胞的侵袭能力较对照组明显降低(P0.05);TGF-β1 inhibitor组细胞的迁移能力较对照组明显降低(P0.01);TGF-β1被抑制后,细胞中的Akt和ARK5蛋白表达量较对照组显著下降(P0.05)。结论HCC组织中ARK5的表达与肿瘤大小、组织分化程度、肿瘤分期之间存在密切联系,这可能是与TGF-β1刺激Akt/ARK5信号转导通路促进了AMMC-7721细胞的增殖、侵袭和转移相关。
[Abstract]:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignant tumors in the world, studies have reported that HBV and HCV HCC infection was the major cause in Europe and some other developed countries or regions, alcoholic liver cirrhosis associated with obesity and nonalcoholic fatty liver disease is mainly caused by the risk of HCC the occurrence and development of malignant tumor is usually occurred during.HCC complex multistep process interaction in vivo by various factors involved in the body is composed of many factors and the interaction of multi pathway results, although at present has been widely used in clinical routine radiotherapy and chemotherapy technology to effectively control the occurrence and development of HCC the prognosis of most patients, but after receiving the therapy is still not optimistic. Targeted therapy is a new discovery, a new way to treat HCC effectively, has become As a hot and difficult content based medical oncology research. The majority of medical workers who are attempting to become a new target for clinical treatment of HCC, in order to lay the foundation for the prognosis of patients with.ARK5 and improve the effective control of HCC (AMPK-related protein kinase 5) abnormal expression is closely related with the occurrence and development of clinical a variety of malignant tumors, and malignant tumor invasion and metastasis. It has been confirmed that ARK5 is highly expressed in a variety of malignant tumors in human tissues, especially in the invasion and metastasis of highly malignant tumors, but its expression in HCC research is relatively small. The Akt (Protein Kinase B) in HCC and how to regulate ARK5 and HCC in the development of the tumor proliferation, apoptosis and invasion mechanism of the process is still less, beta 1 (transforming growth FA TGF- Ctor-beta 1) signaling pathway abnormalities are malignant tumor occurrence, development, invasion, metastasis and other important factors in the process of TGF-, beta 1 signaling pathway abnormalities are the malignant tumor occurrence, development, invasion, metastasis and other important factors in the process. This study was to investigate the expression and significance of ARK5 in patients with HCC tumor tissues, analysis the expression of ARK5 on proliferation and apoptosis of HCC tumor cells, invasion effect, explore tgf- beta 1 and Akt signal activation on the regulation of the expression of ARK5 HCC in tumor cells and its effect on the biological behavior of HCC cells to tumor, clinical diagnosis and target for understanding the mechanism of occurrence and development of HCC and HCC to provide a reliable basis for the purpose of this study is to observe the treatment. ARK5 in hepatocellular carcinoma (hepatocellularcarcinoma, HCC) expression and significance of tumor patients, analysis of the expression of ARK5 on proliferation and apoptosis of HCC tumor cells, invasion. Ring, explore tgf- beta 1 and Akt signal activation on the regulation of the expression of ARK5 HCC in tumor cells and its effect on the biological behavior of HCC cells to tumor, clinical diagnosis and target for understanding the mechanism of occurrence and development of HCC and HCC to provide a reliable basis for treatment. Methods (1) using fluorescence quantitative PCR in 20 cases HCC and ark5mrna expression in adjacent normal tissue samples were measured; ARK5 protein expression detected by using the Westernblot method of HCC tissues and adjacent normal tissues; ARK5 protein expression detected by immunohistochemistry method in the analysis; the expression of ARK5 and HCC with clinicopathological parameters and survival rate the correlation. (2) sirna-ark5 transfected human hepatoma cell line ammc-7721, in order to reduce the expression of ARK5 in the cells; ammc-7721 cells detected by fluorescence quantitative PCR and Westernblot method ark5mrna And the corresponding protein expression; MTT was used to measure the proliferation of transfected cells; annexinv-fitc/pi double staining method to detect the transfection effect on cell apoptosis; invasion detection of transfected cells by transweii chamber assay; cell scratch assay after transfection can influence on cell migration force; detection of Caspase-3 in the transfected cells (activity. 3) tgf- beta 1 inhibitor ly364947 on human hepatocellular carcinoma cell line ammc-7721, in order to reduce the expression of tgf- beta 1 cells, BrdU cell proliferation assay to detect cell proliferation; cell detected by transweii chamber invasion; migration of cell scratch assay; cell Westernblot detection of tgf- beta 1, Akt, the expression of ARK5 protein. Results (1) the expression of ARK5 and related protein mRNA in HCC carcinoma tissues was significantly higher than the expression in adjacent normal liver tissues; the high expression of ARK5 and HCC Tumor size, histological grade, tumor stage was significantly correlated (P0.05); and the patient's age, liver cirrhosis, serum HBsAg, independent of AFP (P0.05); the expression of ARK5 protein can be used as a HCC independent prognosis factors. (2) the expression of ARK5 AMMC-7721 cells in mRNA and protein after transfection of siRNA-ARK5 significantly decreased (P0.05); group siRNA-ARK5 AMMC-7721 cell proliferation activity was significantly lower than that of siRNA-NC group and blank group, siRNA-ARK5 group and the cell growth inhibition rate at 48 h was the highest, compared with the siRNA-NC group (P0.01); 48 h after transfection, the apoptosis rate of cells in siRNA-ARK5 group increased significantly compared with the control group had significant difference (P0.01) group siRNA-ARK5; cell invasion ability decreased significantly, with significant difference compared with the control group (P0.05); compared with the control group, the migration ability of siRNA-ARK5 cells was significantly reduced (P0.01); the activity of Caspase-3 cells in siRNA-ARK5 group Significantly higher than the control group (P0.05) (3) TGF- beta 1 inhibitor Ly364947 on AMMC-7721 cell, TGF- beta 1 group inhibitor cell proliferation activity was significantly lower than the control group (P0.01); group TGF- beta 1 inhibitor cell invasion ability was significantly lower than the control group (P0.05); the migration ability of TGF- beta 1 inhibitor the cell group was significantly lower than the control group (P0.01); TGF- beta 1 is inhibited, the expression of Akt and ARK5 protein were significantly decreased compared to the control group (P0.05). The expression of ARK5 and tumor size. Conclusion HCC tissues, tissue differentiation, there is a close relationship between tumor stage, it is possible to promote the proliferation of AMMC-7721 cells and TGF- beta 1 stimulation of Akt/ARK5 signal transduction pathway, invasion and metastasis.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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