MicroRNA-222促进结直肠癌迁移和侵袭机制及肝癌源性Exosome诱导MSC分化为CAFs的研究

发布时间：2018-02-10 12:36

本文关键词： 迁移和侵袭 miR-222 MST3 Exosome 间充质干细胞肿瘤相关成纤维细胞　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：远处转移是恶性肿瘤患者死亡的主要原因之一,了解其分子机制将有助于筛选出新的判断预后的生物标志物及治疗靶点。本论文以两种常见的消化道肿瘤结直肠癌及肝癌为研究对象,对其可能的转移机制进行了探讨。在论文的第一部分,系统地研究了 miR-222促进结直肠癌迁移和侵袭的作用机制及miR-222及其靶基因的临床意义。通过体外细胞培养的Transwell实验和体内小鼠肿瘤模型研究了 miR-222对结直肠癌细胞迁移和侵袭的影响,通过生物信息学和双荧光素酶报告发现miR-222可特异性靶向MST3的3'UTR,通过细胞功能实验验证了 MST3基因对结直肠癌细胞系迁移和侵袭能力的影响,并初步探讨了 MST3影响结直肠癌迁移和侵袭的可能为通过调节Paxillin磷酸化,而MST3和Paxillin通路的作用机制需进一步深入研究。在临床研究方面,回顾性收集在北京协和医院行手术切除的结直肠癌患者的临床病理资料及随访结果,发现在临床标本中miR-222与MST3的表达成负相关,MST3在结直肠癌瘤旁正常组织中表达较肿瘤组织高,生存分析发现MST3表达阳性患者的无病生存期长于阴性患者,但MST3在肿瘤组织中的表达对预测结直肠癌患者预后的影响仍需进一步研究。研究结果表明miR-222通过调节MST3,在结直肠癌转移中起关键作用。因此,miR-222有成为结直肠癌新型治疗靶标的应用前景。第二部分,初步研究了肝癌HepG2细胞分泌的Exosome对间充质干细胞(Mesenchymal stem cell,MSC)的分化的影响及其相互作用。肿瘤相关的肌成纤维细胞(Cancer-Associated Myofibroblasts,CAF)是肿瘤组织中最常见的非肿瘤细胞类型,在肿瘤的生长、转移、血管新生和肿瘤基质形成中扮演了重要的角色,阻断肿瘤组织中CAF的形成可能会抑制肿瘤生长,为肿瘤治疗提供新的靶点。我国的肝癌通常是在慢性肝炎-肝纤维化的基础上发展而来的,所以CAF等成纤维类的细胞在肝癌中的作用更为显著,而其中CAF的来源并没有一致的观点。我们的研究发现HepG2细胞分泌的Exosome可促进人脂肪来源MSC表达CAF特征性蛋白。通过MTS增殖实验及Transwell实验证明CAF可增强HepG2的增殖、迁移和侵袭能力,并促进HepG2细胞表达上皮间质转化相关基因。因此,肝癌细胞HepG2分泌的Exosome可以诱导MSC分化为CAF,而CAF又可促进HepG2的增殖、迁移和侵袭,为肝癌治疗研究提供了新的靶点及研究方向。本研究对进一步揭示恶性肿瘤转移的机制提供了依据,对于患者预后判断具有一定的指导价值。
[Abstract]:Distant metastasis is one of the leading causes of death in patients with malignant tumors. Understanding its molecular mechanism will help to screen new biomarkers and therapeutic targets for prognosis. In this study, two common gastrointestinal cancers, colorectal cancer and liver cancer, were studied. In the first part of this paper, the possible transfer mechanism is discussed. The mechanism of miR-222 promoting the migration and invasion of colorectal cancer and the clinical significance of miR-222 and its target gene were studied systematically. The effect of miR-222 on colorectal cancer cells was studied by Transwell experiment in vitro and mouse tumor model in vivo. The effects of migration and invasion, The results of bioinformatics and double luciferase report showed that miR-222 could specifically target MST3, and the effect of MST3 gene on the migration and invasion of colorectal cancer cell lines was verified by cell function experiments. The possible mechanism of MST3 affecting the migration and invasion of colorectal cancer is to regulate the phosphorylation of Paxillin, but the mechanism of MST3 and Paxillin pathway needs further study. The clinicopathological data and follow-up results of patients with colorectal cancer undergoing surgical resection in Peking Union Hospital were retrospectively collected. It was found that the expression of miR-222 and MST3 in clinical specimens was negatively correlated with the expression of MST3 in normal tissues adjacent to colorectal cancer. Survival analysis showed that the disease-free survival of patients with positive MST3 expression was longer than that of patients with negative MST3 expression. However, the effect of MST3 expression in tumor tissues on prognosis of colorectal cancer patients needs further study. The results show that miR-222 plays a key role in metastasis of colorectal cancer by regulating MST3. Therefore, miR-222 may become a new type of colorectal cancer. Prospects for the application of therapeutic targets type type. Part 2, The effects of Exosome secreted by HepG2 cells from hepatocellular carcinoma on the differentiation of mesenchymal stem cells (MSCs) and their interaction were studied. The tumor associated myofibroblast, Cancer-Associated Myofibroblast cells (Cafs), is the most common type of non-tumor cells in tumor tissues. Metastasis, angiogenesis and tumor matrix formation play an important role. Blocking the formation of CAF in tumor tissue may inhibit tumor growth. To provide a new target for cancer therapy. Liver cancer in China is usually developed on the basis of chronic hepatitis and liver fibrosis, so fibroblasts such as CAF play a more significant role in liver cancer. We found that Exosome secreted by HepG2 cells can promote the expression of CAF characteristic protein by MSC derived from human fat. CAF can enhance the proliferation of HepG2 by MTS proliferation test and Transwell assay. The ability of migration and invasion, and the expression of genes related to epithelial mesenchymal transformation in HepG2 cells were promoted. Therefore, Exosome secreted by HepG2 could induce the differentiation of MSC into CAF, while CAF could promote the proliferation, migration and invasion of HepG2. This study provides a new target and research direction for the treatment of liver cancer. This study provides a basis for further revealing the mechanism of malignant tumor metastasis, and has a certain guiding value for the prognosis of patients.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735

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