【作者】 赵海恩；

【导师】 范清宇； 裘秀春；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 研究背景和目的：骨肉瘤是青少年中最常见的原发于骨的的恶性肿瘤，骨肉瘤具有很高的全身转移的倾向，大约20%的患者在初次诊断的时候已经发生肺转移，另外还有40%的患者晚期发生远处器官的转移。随着化疗药物的出现和应用，目前骨肉瘤患者的5年生存率已由上世纪70年代之前的10-20%上升至现在的60-80%。但是无论是原发性骨肉瘤还是转移性骨肉瘤，对常规的化疗药物都具有耐药性，肿瘤耐药是骨肉瘤患者保肢手术治疗失败及发生转移的主要原因，而肿瘤发生转移是骨肉瘤患者死亡的主要原因。转移性肿瘤对治疗不敏感的主要原因是药物抵抗。研究发现，发生肺转移的骨肉瘤细胞表现出了和原发性肿瘤细胞完全不同的生物学特性，发生转移后的肿瘤细胞群在细胞的倍增速度、酶表达谱、染色体组型和化疗药物的敏感性方面都不同于其原发的肿瘤细胞群。这就解释清楚了对原发肿瘤有效的化疗方案，为什么却对转移性肿瘤丝毫不起作用。恶性肿瘤最后在人体内形成转移病灶需要经过很多重要的步骤。要能够从分子水平上控制肿瘤的转移，首先需要对肿瘤的转移机制有彻底的认识，通过控制肿瘤转移的各个步骤，进而防止肿瘤发生转移。尽管经过的大量的基础研究和临床试验，骨肉瘤的转移机制仍然不是很清楚。对骨肉瘤转移的生物学机制进行彻底研究将会为治疗骨肉瘤提供革命性的理论基础。microRNA作为基因的一种表现形式，是否在骨肉瘤发生转移的过程中发挥作用以及以何种形式发挥作用，引起了学者的关注。因此，本实验旨在通过比较高、低不同转移能力的骨肉瘤细胞中microRNA的表达水平的差异，并通过瞬时转染microRNA的方法对细胞进行干预，观察干预后肿瘤细胞的生长及转移特性，以探索瞬时转染microRNA对骨肉瘤细胞生物学性质的即刻效应，为更好的明确骨肉瘤的转移机制提供理论依据和实验指导。研究方法：首先比较高、低转移能力骨肉瘤细胞中miR-183及Ezrin蛋白的表达差异，根据表达水平的差别对骨肉瘤细胞转染外源性miR-183，建立不同表达水平miR-183的骨肉瘤细胞模型。然后采用MTT、流式细胞仪、细胞划痕实验、transwell实验、免疫细胞化学、westernbloting等实验方法对细胞的生长特性和形态学表现进行研究，以评估miR-183在不同转移能力细胞中的表达，miR-183对细胞蛋白表达水平的影响和细胞转移能力的影响。最后通过相关的统计学分析，以明确miR-183表达水平与Ezrin蛋白的表达量、肿瘤的侵袭和转移能力之间的相关性。研究结果：1.无论是在骨肉瘤细胞还是在骨肉瘤组织中，高转移性骨肉瘤中Ezrin蛋白的表达量高于低转移性骨肉瘤中的表达；而实时定量PCR研究发现，高转移性骨肉瘤细胞中miR-183的表达量显著低于高转移性细胞中的表达量。2.通过瞬时转染外源性miR-183可以使高转移性骨肉瘤细胞F5M2中miR-183的表达量显著升高，成功建立不同表达水平miR-183的骨肉瘤细胞模型。3.MTT、流式细胞仪、细胞划痕实验、transwell等实验结果表明外源性过表达miR-183的F5M2细胞转移能力显著下降，呈剂量依赖关系；miR-183对F5M2细胞的增殖和凋亡能力无影响。4.免疫细胞化学、westernbloting等实验结果表明外源性过表达miR-183的F5M2细胞中Ezrin蛋白的表达量下降，呈剂量依赖关系。结论：高转移性骨肉瘤细胞F5M2中miR-183的表达量较低转移性骨肉瘤F4中表达量较低，而高转移性肿瘤中Ezrin蛋白的表达量显著高于低转移性肿瘤。通过转染外源性miR-183可以显著提高miR-183在F5M2的表达量，降低Ezrin蛋白表达。miR-183的表达量提高不仅会使F5M2中Ezrin蛋白的表达量降低，而且可以使肿瘤的侵袭和迁移能力显著抑制，而增殖和凋亡特性无明显变化。相关性研究发现，Ezrin蛋白的表达量和骨肉瘤细胞的转移潜能存在正相关。这些研究结果提示通过提高miR-183在高转移性骨肉瘤F5M2中的表达量，可以抑制Ezrin蛋白的表达，从而抑制肿瘤转移潜能。miR-183抑制骨肉瘤转移的这一机制还有待于进一步的实验验证，且为深入的基础研究和潜在的临床应用开辟了新途径。更多还原

【Abstract】 Background and Objective:Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. The estimated incidence rate worldwide is4/million/year, with a peak incidence at the age of15-19years. In OS there is a high tendency to metastatic spread. Approximately20%of patients present with lung metastases at initial diagnosis and, additionally, in40%of patients metastases occur at a later stage. Eighty percent of all metastases arise in the lungs, most commonly in the periphery of the lungs, and exhibit resistance to conventional chemotherapy. The5-year survival rate for OS patients with metastases is20%compared to65%for patients with localised disease and most deaths associated with OS are the result of metastatic disease.Metastasis is considered to be the final though most critical step in tumorigenesis of malignant tumours. It has been shown that OS lung metastases display a biological behaviour different from the primary tumours. Metastases are comprised of cell clones that differ from primary tumours with respect to ploidy, enzyme profile, karyotype and chemosensitivity. Therapeutic regimens that target primary tumours are therefore unlikely to be successful in the treatment of metastatic disease.The metastatic cancer cells subsequently complete many steps to metastasis. Over the last decade, much research has been performed to try to unravel the biology of OS metastasis and many (pre)clinical studies have attempted to discover new treatment options for metastatic OS. However, the exact metastatic mechanism of OS remains unclear. Further study about the molecular mechanism of OS will provide novel treatments for therapy in metastatic OS.miRNA expression profiling analyses have reported a general dysregulation of miRNA expression in all tumors. In this study we will outline the biological and molecular roles of miRNAs in OS and metastasis by highlighting the most promising advances for the use of miRNAs in the clinical setting. The aim of this study is to explore the biology of metastatic OS cells and how these may contribute to designing a metastasis directed treatment for OS.Methods:Firstly, we compared the differences of miR-183and Ezrin expression in high-metastatic human OS F5M2and low-metastatic human OS F4cells. Functional analysis was then carried out by transfection of miR-183mimics or inhibitors into the high-metastasis OS F5M2cell line with low endogenous miR-183expression. The results of the transfection were subsequently assessed on cell viability pattern by proliferation and apoptosis assay, cell migration by transwell insert and wound healing assay, gene expression alterations by real-time PCR and protein levels by western blotting and immunocytochemistry (ICC). At last we study the relationship between the miR-183level and the metastatic potential of OS.Results:1. The expression level of Ezrin in high metastatic OS was significantly higher than that of in low metastatic OS. There is a same tendency either in cells or in samples. Real-time PCR demonstrated that miR-183expression in high metastatic F5M2cells was significantly lower than in low metastatic F4cells.2. It showed that miR-183was significantly over-expressed in F5M2cells after transfection with the miR-183mimics. In this study, we successfully established OS cell models with different expression of miR-183. 3. Transwell insert and wound healing assay demonstrated that ectopic expression of miR-183inhibited F5M2cell migration and invasion potential in vitro. The results of flow cytometry and MTT assay showed that there was no difference on apoptosis and proliferation between F5M2cells untreated or transfected with miR-183mimics.4. Western blotting and ICC showed that the expression of Ezrin in F5M2cells decreased markedly after transfection with miR-183mimics, compared with cells untreated or treated with NC. There was an inverse correlation between Ezrin production and miR-183levels.Conclusion:Overexpression of miR-183mainly inhibited the migration and invasion of F5M2cells, not proliferation and apoptosis. Ezrin expression was positively correlated to migration and invasion of OS cells. It is concluded that alteration of miR-183might act as a significant inhibitory factor in regulate cell migration and invasion targeting the downregulation of Ezrin expression. To our knowledge, this is the first in vitro study to regulate metastasis and progression of OS, by upregulation of miR-183to target the expression of Ezrin in F5M2cells. Our study might provide an important avenue for further analysis in vivo with the aim to develop a new potential diagnostic and therapeutic targets for the screening and treatment of high metastatic OS. Further studies are required to fully understand the regulation mechanisms of miR-183and Ezrin in OS in vitro and in vivo.更多还原