线粒体融合蛋白2在肝细胞肝癌中作用机制的研究

发布时间：2018-06-14 13:36

本文选题：线粒体融合蛋白2 + 线粒体　；参考：《浙江大学》2016年博士论文

【摘要】：目前,肝细胞性肝癌(Hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤疾病,占我国恶性肿瘤死亡原因的第二位,肝癌早期往往不易发现,导致多数患者不能接受手术治疗,仍缺乏有效的治疗方法。肿瘤的形成多与过度活化的细胞增殖以及受抑制的细胞凋亡相关。目前的研究热点及针对肝癌的治疗方法也主要是通过各种手段抑制肿瘤细胞的异常增殖、促进癌细胞凋亡。细胞增殖抑制基因(hyperplasia suppressor gene, HSG)是利用差异显示技术得到的一个与高血压相关的新基因,可以抑制血管平滑肌细胞的增殖、促进其凋亡,并在后续研究中发现HSG蛋白也参与调节线粒体融合,因此也被称为线粒体融合蛋白2(MFN2)。随着研究的深入,进一步发现MFN2跟很多肿瘤的恶性生物学特征相关,提示MFN2可能与肿瘤发生发展密切相关；同时,也有部分研究发现,MFN2可以促进癌细胞凋亡,因此也逐渐成为肿瘤研究的热点。第一部分MicroRNA-761在肝癌中低表达并通过抑制线粒体融合蛋白2表达影响肝癌细胞生长目的：线粒体融合蛋白2(mitofusin-2, MFN2)是锚定在线粒体外膜的调节蛋白,是线粒体融合蛋白家族的重要成员,负责调节相邻线粒体外膜的融合,对维持线粒体分裂、融合的平衡以及线粒体的稳态起到重要作用。越来越多的研究显示,MFN2可能为一个新的抑癌基因,并且在恶性肿瘤的发生、发展中起到重要的作用。MFN2在多个肿瘤中低表达的原因,以及是否受到miRNAs的调节,并没有被阐明。本实验旨在阐明肝癌细胞中MFN2的转录后调节机制。方法：通过网站(target scan)预测分析得到MFN2基因的潜在miRNA-miRNA-761,运用荧光素酶双报告实验确认能其能直接调节MFN2基因；通过qRT-PCR分析其在肝癌组织中的表达量,以及其表达量与MFN2表达量的关系。外源性导入miRNA-mimic和inhibitor,分析其对肝癌细胞增殖、细胞凋亡、线粒体膜电位、活性氧水平以及细胞侵袭迁移的影响；构建裸鼠原位肝癌移植模型,阐明miRNA在体内对肝癌细胞增殖和转移的作用。结果：MiRNA-761可以直接作用于MFN2的3'-UTR区,并可在mRNA和蛋白水平调节其表达；MiRNA-761在肝癌组织中低表达,并且表达量与MFN2呈负相关性；MiRNA-761抑制剂可以降低线粒体膜电位,促进细胞凋亡,并在体内外抑制肝癌细胞的增殖和转移。结论：MiRNA-761抑制剂可以通过上调MFN2的表达,损伤线粒体功能,从而导致肝癌细胞增殖受到抑制,并促进肝癌细胞凋亡。第二部分线粒体融合蛋白2表达相关基因神经导航分--子-3与肝癌预后的相关性分析目的：阐明与MFN2表达相关的基因,进一步研究MFN2的作用机制；通过生存分析找寻肝癌患者预后的相关分子标志,为肝癌的治疗提供潜在的治疗靶点。方法：通过表达谱芯片分析过表达MFN2后,肝癌细胞中mRNA水平的变化；通过免疫组织化学法检测肝癌组织中神经导航分子3(Neuron navigator 3, NAV3)的含量；联合MFN2及NAV3对肝癌患者进行生存分析；检测NAV3过表达后对肝癌细胞增殖、转移、侵袭、凋亡的影响。结果：表达谱芯片显示NAV3表达量与MFN2密切相关。NAV3在肝癌组织中的表达量与患者的预后、肿瘤的大小、肿瘤分化以及AJCC分期有关。联合NAV3和MFN2进行生存分析可以提供更好的预测价值；在肝癌细胞系中过表达NAV3可以抑制细胞增殖、转移侵袭、促进细胞凋亡,并在体内抑制裸鼠皮下移植瘤的生长。结论：本实验证明,NAV3表达量与MFN2相关,并在肝癌中起到重要作用。低表达NAV3的患者预示着较差的预后,NAV3可以作为肝癌患者的预后分子标记,也是肝癌的潜在治疗靶点。
[Abstract]:At present, Hepatocellular carcinoma (HCC) is the most common malignant tumor in China, which accounts for second of the causes of the death of malignant tumors in China. The early liver cancer is often difficult to detect in the early stage of the liver cancer, which causes most patients to be unable to receive surgical treatment and still lacks effective treatment methods. Hyperplasia suppressor gene (HSG) is a new basis for the use of differential explicit techniques to inhibit the proliferation of cancer cells and promote the apoptosis of cancer cells. Because it can inhibit the proliferation of vascular smooth muscle cells and promote its apoptosis, it is found that HSG protein is also involved in regulating mitochondrial fusion in subsequent studies. Therefore, it is also known as mitochondrial fusion protein 2 (MFN2). With the further research, it is found that MFN2 is associated with the malignant biological characteristics of many tumors, suggesting that MFN2 may be associated with the occurrence of tumors. At the same time, some studies have found that MFN2 can promote the apoptosis of cancer cells, and therefore gradually become a hot spot in cancer research. The first part is the low expression of MicroRNA-761 in liver cancer and the effect of inhibiting the expression of mitochondrial fusion protein 2 to affect the growth of hepatoma cells: the mitofusin-2 (MFN2) is anchored. The regulatory protein of the mitochondrial membrane, an important member of the mitochondrial fusion protein family, is responsible for regulating the fusion of adjacent mitochondrial membrane and plays an important role in maintaining mitochondrial division, fusion balance and the homeostasis of mitochondria. More and more studies have shown that MFN2 may be a new tumor suppressor gene and in the occurrence of malignant tumor. This study aims to elucidate the post transcriptional regulation mechanism of MFN2 in hepatoma cells. Methods: the potential miRNA-miRNA-761 of the MFN2 gene was obtained through the web site (target scan) prediction analysis and the use of luciferase double. The experiment confirmed that the MFN2 gene could be directly regulated, and the expression of its expression in liver cancer tissue was analyzed by qRT-PCR and the relationship between its expression and the expression of MFN2. Exogenous miRNA-mimic and inhibitor were introduced, and the effects on the proliferation, apoptosis, mitochondrial membrane potential, active oxygen level and cell invasion and migration of hepatoma cells were analyzed. A model of orthotopic liver cancer transplantation in nude mice was constructed to elucidate the effect of miRNA on the proliferation and metastasis of hepatoma cells in vivo. Results: MiRNA-761 can directly act on the 3'-UTR region of MFN2 and regulate its expression at the level of mRNA and protein; the expression of MiRNA-761 in the liver cancer tissues is low, and the amount of the expression is negatively correlated with the MFN2; MiRNA-761 inhibitors It can reduce the mitochondrial membrane potential, promote cell apoptosis and inhibit the proliferation and metastasis of hepatoma cells in vivo and in vitro. Conclusion: MiRNA-761 inhibitor can inhibit the proliferation of hepatoma cells by up regulation of MFN2 expression and damage mitochondrial function, and promote the apoptosis of hepatoma cells. The second part of mitochondrial fusion protein 2 expression phase Correlation analysis of gene neuronavigation score - sub-3 and prognosis of hepatocellular carcinoma Objective: to elucidate the genes associated with MFN2 expression, to further study the mechanism of MFN2, to find the molecular markers of the prognosis of liver cancer by survival analysis, and to provide potential therapeutic targets for the treatment of liver cancer. Changes in the level of mRNA in hepatoma cells after MFN2; the content of neuronavigation molecule 3 (Neuron navigator 3, NAV3) in liver cancer tissue was detected by immunohistochemistry; the survival analysis of patients with hepatocellular carcinoma combined with MFN2 and NAV3; the effects of NAV3 overexpression on the proliferation, metastasis, invasion and apoptosis of hepatoma cells. Results: expression profiles The expression of NAV3 expression and MFN2 closely related to the expression of.NAV3 in liver cancer tissues is related to the prognosis of the patients, the size of the tumor, the differentiation of the tumor and the staging of AJCC. The survival analysis of NAV3 and MFN2 can provide better predictive value; the overexpression of NAV3 in the hepatoma cell line can inhibit cell proliferation, metastasis and invasion. Apoptosis, and inhibit the growth of subcutaneous transplanted tumor in nude mice in vivo. Conclusion: this experiment shows that the expression of NAV3 is associated with MFN2 and plays an important role in liver cancer. The patients with low expression of NAV3 indicate a poor prognosis. NAV3 can be used as a molecular marker for the prognosis of liver cancer, and also a potential target for the treatment of liver cancer.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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