细胞表型转化促进肿瘤转移的新机制研究

发布时间：2018-07-21 16:36

【摘要】：恶性肿瘤生长速度快,具有侵袭浸润能力,且常有远处转移。肿瘤转移是指肿瘤细胞从原发部位脱离,经血管、淋巴道或体腔等途径,最终到达机体其他部位继续生长增殖的过程。肿瘤转移是罹患恶性肿瘤的患者高死亡率的罪魁祸首,因此人们一直致力于肿瘤转移机制的研究。细胞上皮样表型与间质样表型之间的转化过程与很多生理病理过程密切相关,如胚胎发育、肿瘤转移等。肿瘤细胞经历上皮-间质转化(epithelial-mesenchymal transition,EMT)时,细胞间粘附连接性降低,极性逐渐丧失转变为间质样状态,细胞更具移动能力,有利于肿瘤早期侵袭转移。在肿瘤转移的晚期,肿瘤细胞经历间质-上皮转化(mesenchymal-epithelial transition,MET),重塑上皮样表型,促进转移灶形成。可以认为,EMT和MET在肿瘤转移的不同阶段发挥各自不同的作用。细胞表型转化的一个重要标志是细胞粘附分子E-钙粘素(E-cadherin)表达水平的变化,该分子被视为上皮表型的标志物。此外,一些间质样表型的标志蛋白,如波形蛋白(Vimentin)、N-钙粘素(N-cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达水平的变化也可以反映细胞是否发生细胞表型的转变。作为Toll样受体炎症信号通路的关键衔接蛋白,髓样分化因子88(Myeloid differentiation factor 88或Myeloid differentiation primary response 88,简称My D88)发挥着重要的信号转导作用。已有很多研究证实My D88在多种肿瘤组织中表达升高,并通过炎症反应途径促进肿瘤发生发展。最近的研究发现,除了通过Toll样受体介导的炎症反应通路促进肿瘤发展外,My D88还可以通过非炎症途径调控肿瘤的发展。我们前期通过对110例肝细胞肝癌组织进行免疫组织化学染色,发现My D88在癌组织中的表达高于相应的癌旁组织,而My D88的高表达和肝细胞肝癌患者的不良预后密切相关;此外,前期研究结果显示My D88高表达可以促进肿瘤的生长与转移。因此,探讨肿瘤细胞中My D88高表达如何通过非炎症途径促进肿瘤转移是本项研究需要解决的重点问题。骨桥蛋白(osteopontin,OPN)通常被认为是一种分泌型磷酸化糖蛋白,研究者们也确实发现分泌到细胞外的OPN生物活性十分多样。有趣的是,人们后来发现OPN并不只是一种分泌型蛋白,在细胞核、细胞膜等均发现OPN的表达分布,并参与了细胞的功能调节。近年来,OPN在肿瘤发生发展过程中发挥的作用日益引起人们关注。在肝癌、乳腺癌、胃癌和肺癌等多种癌组织中均发现OPN表达增加,并且在一些肿瘤转移病人血液中亦出现高水平表达。OPN促进肿瘤转移可能是通过参与调节上皮间质表型转变而实现的。因此,在肿瘤转移研究方面,对OPN的研究前景广阔。本课题旨在研究My D88与OPN两种分子在肿瘤细胞中的表达,探讨两种分子对肿瘤细胞表型转化及肿瘤转移的作用与机制,并对其临床意义加以阐释。在前半部分,我们首先利用过表达慢病毒和sh RNA慢病毒改变My D88的表达水平,通过检测细胞表型转化相关标志蛋白在蛋白质水平和细胞内定位的变化,以及对EMT相关转录因子的检测,研究My D88在上皮、间质表型转化中的调控作用,并在肝癌临床标本中加以验证。之后通过体外实验检测肿瘤干细胞相关标志分子和体内成瘤实验发现,My D88的高表达不但诱导发生EMT,对肿瘤细胞干性特征的表达也非常关键。之后我们探讨My D88促进EMT的内在机制。通过激酶活性检测、免疫共沉淀实验、细胞表型标志蛋白检测和体内肿瘤转移等实验,发现My D88通过与PI3-K的p85调节亚基直接结合,激活PI3-K/Akt/GSK-3β/Snail信号通路,促进EMT和肿瘤转移。最后,我们检测了肝癌细胞株和肝癌临床标本中My D88和p-Akt的表达情况,对其相关性和两者对肿瘤患者预后的影响进行了统计学分析,提示My D88表达水平和Akt磷酸化水平升高促进肝癌转移,并且综合分析My D88和p-AKT对肝癌预后判断具有更好价值。在后半部分,我们主要检测OPN在肿瘤细胞中的表达分布,研究不同表达形式的OPN对细胞表型转化的作用,并对转移灶中OPN转入细胞核表达的机制进行探讨。我们首先检测多种肿瘤细胞中OPN的表达定位,发现在肿瘤细胞除了表达经典的分泌型OPN,还表达仅分布于细胞核的OPN。紧接着,利用sh RNA慢病毒和过表达慢病毒调控OPN的表达水平,通过检测细胞表型转化相关标志分子在蛋白质水平和细胞内定位的变化,以及细胞表型转化相关转录因子的变化,研究不同形式OPN对细胞表型转化的作用。我们发现分泌型OPN和细胞核型OPN作用不同:分泌型OPN促进肿瘤细胞发生EMT,而细胞核型OPN诱导MET发生。我们前期发现细胞核型OPN诱导MET发生的过程中,AKT1/mi R-429/ZEB轴起关键作用。在此我们继续探讨细胞核型OPN调控AKT1表达的机制。通过双荧光素酶报告基因实验、免疫荧光双染色、免疫共沉淀等实验,发现细胞核型OPN直接结合HIF2α,阻断HIF2α在转录水平对AKT1的表达抑制,这种作用使得mi R-429表达上调,从而可以作用于ZEB诱导细胞发生MET。我们前期通过动物体内实验和检测肝癌临床标本发现,在原发灶中OPN主要表达在细胞质,诱导EMT促进肿瘤转移扩散;在转移灶中OPN转入细胞核中表达并诱导MET,促进癌细胞定植形成转移灶。之后,我们探讨了转移灶中诱发OPN转入细胞核内的机制。通过皮下肿瘤转移实验,收集不同时期肺组织用于抗体芯片检测,我们发现Leptin、FGFa和VEGF三种变化明显的细胞因子。刺激细胞并检测OPN定位变化,筛选出诱发OPN转入细胞核分布的细胞因子VEGF。之后通过检测OPN修饰水平的改变、激酶抑制、抗体中和等实验,我们证明VEGF通过KDR/PLCγ/PKC路径促使OPN转入细胞核,进而诱导MET和转移灶形成。本课题通过对My D88与OPN两种分子的研究,阐述了细胞表型转化(EMT和MET)促进肿瘤转移的新机制。我们证明了My D88可以在不依赖Toll受体的情况下,直接与PI3-K的p85亚基结合,启动下游信号通路,诱导细胞发生EMT,促进肿瘤生长和转移;我们揭示了肿瘤细胞中细胞核型OPN的表达分布、不同表达形式的OPN对细胞表型转化的调控作用,并发现转移灶微环境中细胞因子的变化促使OPN转入细胞核表达,通过诱发MET促进肿瘤细胞定植和形成转移灶。这些研究或可为肿瘤转移的研究提供全新的依据。
[Abstract]:Malignant tumor grows fast, has invasion and invasion ability, and often has distant metastasis. Tumor metastasis refers to the process of tumor cells from the original site, through the blood vessels, lymphatic channels or body cavity, and finally to the other parts of the body to continue to grow and proliferate. Tumor metastasis is the culprit of high mortality in patients with malignant tumor. The transformation process between the epithelioid and interstitial like phenotype is closely related to many physiological and pathological processes, such as embryo development, tumor metastasis, and so on. When the tumor cells undergo epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT), the intercellular adhesion connectivity is reduced and the polarity is reduced. In the late stage of tumor metastasis, the tumor cells undergo interstitial epithelial transformation (mesenchymal-epithelial transition, MET), reshape the epithelioid phenotype and promote metastatic foci in the late stage of tumor metastasis. It can be considered that EMT and MET are occurring at different stages of tumor metastasis. One of the important markers of cell phenotypic transformation is the change in the expression level of cell adhesion molecule E- cadherin (E-cadherin), which is considered as a marker of epithelial phenotype. In addition, some markers of interstitial like phenotypes, such as vimentin (Vimentin), N- cadherin (N-cadherin), and alpha smooth muscle actin (alpha -smoo) The changes in the expression level of th muscle actin, alpha -SMA also reflect cell phenotype transformation. As the key cohesive protein of the inflammatory signaling pathway of the Toll like receptor, the myeloid differentiation factor 88 (Myeloid differentiation factor 88 or Myeloid differentiation primary response 88, for short), plays an important signal turn. Guidance. Many studies have shown that My D88 is elevated in a variety of tumor tissues and promotes the development of tumors through an inflammatory response pathway. Recent studies have found that My D88 can also regulate tumor development through non inflammatory pathways through the Toll like receptor mediated inflammatory pathway. After immunohistochemical staining of 110 hepatocellular carcinoma tissues, it was found that the expression of My D88 in the cancer tissues was higher than that of the corresponding para cancerous tissue, and the high expression of My D88 was closely related to the poor prognosis of the liver cancer patients. In addition, the earlier results showed that the high expression of My D88 could promote the growth and metastasis of the tumor. Therefore, the study of the high expression of My D88 can promote the growth and metastasis of the tumor. The high expression of My D88 in tumor cells how to promote tumor metastasis through non inflammatory pathways is the key problem to be solved in this study. Osteopontin (OPN) is generally considered to be a secretory phosphorylated glycoprotein, and researchers do find that the biological activity of OPN secreted out of cells is very diverse. To find that OPN is not only a secretory protein, the expression distribution of OPN is found in the cell nucleus and cell membrane, and the function regulation of the cells is involved. In recent years, the role of OPN in the development of tumor has attracted more and more attention. The expression of OPN is increased in many kinds of cancer tissues such as liver cancer, breast cancer, gastric cancer and lung cancer. There is also a high level of expression of.OPN in the blood of some patients with tumor metastasis, which may be achieved by regulating the phenotype of epithelial mesenchymal transition. Therefore, the study of tumor metastasis has a broad prospect for the study of OPN. The purpose of this study is to study the expression of two molecules of My D88 and OPN in tumor cells, and explore two In the first half, we use the expression of lentivirus and sh RNA lentivirus to change the expression level of My D88 by detecting the changes in the protein level and intracellular localization of the egg white by the cell phenotype transformation. And the detection of EMT related transcription factors, study the regulatory role of My D88 in epithelial and interstitial phenotypic transformation, and verify it in the clinical specimens of liver cancer. After the detection of tumor stem cell related markers and in vivo tumorigenesis experiments in vitro, the high expression of My D88 not only induces the occurrence of EMT, but also the dry character of the tumor cells. The expression is also crucial. Then we explore the intrinsic mechanism of My D88 to promote EMT. Through the experiments of kinase activity detection, immunoprecipitation experiment, cell phenotype marker protein detection and tumor metastasis in vivo, we found that My D88 activates PI3-K /Akt/GSK-3 beta /Snail signaling pathway through the p85 regulated subunit of PI3-K, and promotes EMT and tumor turn. Finally, we detected the expression of My D88 and p-Akt in the liver cancer cell lines and the clinical specimens of liver cancer, and analyzed the correlation and the effect of the two on the prognosis of the tumor patients. It was suggested that the expression level of My D88 and the increase of Akt phosphorylation level promoted the metastasis of liver cancer, and the prognosis of the liver cancer was judged by My D88 and p-AKT. It is of better value. In the second half, we mainly examine the expression distribution of OPN in tumor cells, study the effect of different expressions of OPN on cell phenotype transformation, and discuss the mechanism of OPN transfer to the nuclear expression in the metastases. We first detected the expression of OPN in various tumor cells, and found that the tumor cells were found to be removed from the tumor cells. The expression of the classical secretory OPN, and the expression of the OPN. only distributed in the nucleus, using the SH RNA lentivirus and the overexpression of lentivirus to regulate the expression level of OPN, the changes in the protein level and intracellular localization of the cell phenotype transformation markers and the changes of the related transcription factors of the cell phenotype transformation are studied. The effect of different forms of OPN on cell phenotype transformation. We have found that secretory OPN and cell nuclear type OPN are different: secretory OPN promotes EMT, and cell nuclear OPN induces MET. We found that AKT1/mi R-429 /ZEB axis plays a key role in the process of cell nuclear OPN induced MET. The mechanism of AKT1 expression by cytoplasmic OPN regulation. Through double luciferase reporter gene experiment, immunofluorescence double staining and immunoprecipitation experiments, it was found that the cell nuclear OPN was directly combined with HIF2 alpha, blocking the inhibition of the expression of HIF2 alpha at the transcriptional level of AKT1. This effect made the MI R-429 table up regulation, which could be used to induce MET in ZEB induced MET. We found that OPN was mainly expressed in the cytoplasm and induced EMT to promote the metastasis and diffusion of tumor in the primary foci, and OPN was transferred into the nucleus to express and induce MET in the metastatic foci, and promote the metastasis of cancer cells to form the metastasis. After that, we explored the induction of OPN into the nucleus in the metastases. Internal mechanism. Through the subcutaneous tumor metastasis experiment, we collect different period lung tissues for antibody chip detection. We found three kinds of cytokine that have obvious changes in Leptin, FGFa and VEGF, stimulate the cell and detect the change of OPN location, and screen the change of the OPN modification level after screening the cytokine VEGF. that induces the transfer of OPN to the nucleus distribution. Kinase inhibition, antibody neutralization and other experiments, we have demonstrated that VEGF induces OPN into the nucleus through the KDR/PLC gamma /PKC pathway, and then induces the formation of MET and metastatic foci. This topic illustrates the new mechanism of cell phenotype transformation (EMT and MET) to promote tumor metastasis through the study of two molecules of My D88 and OPN. In the case of the receptor, it combines with the p85 subunit of PI3-K directly to initiate the downstream signal pathway, induce the cell to develop EMT, promote the growth and metastasis of the tumor; we reveal the expression distribution of the cell nuclear OPN in the tumor cells, the regulation of the different expressions of OPN on the cell phenotype transformation, and the change of the cytokines in the microenvironment of the metastases. It promotes the transfer of OPN into the nuclear expression, and promotes tumor cell colonization and metastasis by inducing MET. These studies may provide a new basis for the study of tumor metastasis.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-37

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