【作者】 张翼；

【导师】 蔡林；

【作者基本信息】 武汉大学 ， 外科学， 2014， 博士

【摘要】 骨肉瘤是最常见的原发性恶性骨肿瘤,以产生不成熟的骨及骨样组织为基本特点。75%的病人年龄在10-30岁,恶性程度高,易早期局部侵袭或远处转移,预后差,是危害青少年健康的主要肿瘤性疾病之一。目前骨肉瘤的致病基因和发病机制研究成为骨肉瘤基础研究中的重点和热点,这些研究可以为临床实践中早期诊断和治疗骨肉瘤发挥重要的指导作用,以后也将成为骨肉瘤诊断和治疗的关键。近十余年以来,研究人员发现microRNA(miRNA)广泛参与原核和真核生物体内基因表达和功能调节。目前研究结果证实,miRNA广泛参与细胞内基因转录调节、信号转导调控,与炎症、肿瘤、内分泌疾病的发生、发展密切相关。miRNA是基因表达和蛋白翻译阶段重要的内源性调节RNA,通过特异性改变靶基因表达水平和功能状态,在肿瘤的发生过程中起到关键的调控作用。本文以表阿霉素(Epirubicin, EPI)抗骨肉瘤过程为基本研究模型,首先确定并量化表阿霉素抑制骨肉瘤增殖的效果,以miRNA表达谱芯片寻找在表阿霉素抗骨肉瘤过程中差异性表达的miRNA,并确认这些miRNA的表达水平。其次根据这些差异性miRNA的功能状态,利用外源性miRNA模拟物在体外研究中有目的性的改变这些miRNA的表达水平,同时联合使用表阿霉素,检测该miRNA在骨肉瘤细胞中的表达水平,以确认其在表阿霉素抗骨肉瘤过程中的作用。最后,我们利用体外miRNA表达调控技术,调节目的miRNA表达,检测其下游可能的多种mRNA表达水平,并检测骨肉瘤细胞凋亡和周期变化,从而确定目的miRNA在表阿霉素抗骨肉瘤过程中的作用,并确定靶基因蛋白在表阿霉素抗骨肉瘤过程中的表达变化和作用。第一部分miRNA在表阿霉素抗骨肉瘤过程中的差异性表达目的：研究表阿霉素处理后骨肉瘤细胞中miRNA表达谱变化,并对其变化趋势进行确证,寻找感兴趣的miRNA以进行功能学研究。方法：首先利用CCK-8法检测表阿霉素对骨肉瘤MG-63和SAOS-2细胞增殖抑制作用,观察表阿霉素处理后骨肉瘤细胞形态学变化。其次应用miRNA表达谱芯片检测表阿霉素处理前后骨肉瘤细胞中miRNA表达水平,寻找出差异性表达的miRNA.然后通过生物信息学研究和现有文献总结,选择部分差异性miRNA作为感兴趣的miRNA。最后以实时定量PCR法确证骨肉瘤中感兴趣miRNA在表阿霉素处理前后差异性表达,为进行下一步功能学研究提供基础。结果：1μg/ml及以上浓度表阿霉素可以明显降低骨肉瘤细胞增殖,且这种抑制作用具有浓度依赖性。以lug/ml表阿霉素处理骨肉瘤MG-63和SAOS-2细胞后,细胞形态发生明显改变,骨肉瘤细胞miRNA表达谱发生显著改变,筛选后共发现40个差异性miRNA,其中包括16个上调性表达,24个下调性表达。miR-302b在表阿霉素处理骨肉瘤细胞后呈显著上调性表达；根据生物信息学研究和现有文献查阅,miR-302b可能在骨肉瘤细胞凋亡和周期分布中发挥重要作用。结论：表阿霉素可以通过改变骨肉瘤中miRNA表达谱抑制细胞增殖,共有40个可能的差异性miRNA参与调节这一过程,其中上调性表达miR-302b可能具有重要功能学意义。第二部分miR-302b在表阿霉素抗骨肉瘤过程中功能和作用机制目的：上调骨肉瘤细胞中miR-302b表达水平,研究其单独或与表阿霉素协同抗骨肉瘤作用。方法：利用Lipo2000和miR-302bmimics对骨肉瘤MG-63和SAOS-2细胞进行转染,转染之后通过实时定量PCR检测骨肉瘤细胞在不同因素处理后miR-302b表达水平。在不同因素处理骨肉瘤细胞后,以CCK-8法检测骨肉瘤细胞增殖率；以AnnexinV-PE和7-AAD双标法检测骨肉瘤细胞凋亡率；以碘化丙啶(PI)染色法确定骨肉瘤细胞周期分布变化；以酶切后产物比色法检测Caspase-3活性变化；以Western-blot法检测骨肉瘤细胞内凋亡和周期相关调控蛋白表达水平。结果：以miR-302b模拟物转染骨肉瘤细胞后发现miR-302b表达上调可以明显抑制骨肉瘤细胞增殖,且与表阿霉素具有协同作用；miR-302b表达上调可以明显促进骨肉瘤细胞凋亡,并促使细胞周期G1阻滞,抑制骨肉瘤细胞增殖；miR-302b通过激活Caspase-3,降低AKT、pAKT、Bcl-2、Cyclin D1、CDK2/4并提高Bim表达诱导骨肉瘤细胞凋亡和周期阻滞。结论：上调miR-302b表达可以调节多种凋亡和周期调控蛋白表达水平,从而促进骨肉瘤细胞凋亡且进入细胞周期G1阻滞,抑制骨肉瘤细胞体外增殖,介导表阿霉素抗骨肉瘤过程。更多还原

【Abstract】 Osteosarcoma (OS) is the most common aggressive sarcoma of the bone characterized by producing immature bone or osteoid tissue. Seventy-five percent of patients are diagnosized at15-30years old. Osteosarcoma is highly aggressive and at risk of local relapse or distant metastasis. The prognosis for the OS patients is not good so it is a crippling disease for adolescents and young adults. Thepathogenic genes and pathogenesis of OS are important and key part of the basic research, which could be an important bio-marker for the OS early diagnosis and treatment to improve OS survival.Recently, the investigators find that microRNA could play important role in the target gene regulation. It’s also known that miRNAs and their target genes have the potential to regulate various critical biological processes, including the gene expression, cell signaling, which is highly associated with the inflammation, tumor and endocrine disease. The effects of miRNAs on the gene transcription and protein expression could result in the tumorigenesis.We select the anti-OS effect of epirubicin (EPI) as the research model in the miRNA functions. First, we investigate the inhibitory effect of epirubicin on the OS proliferation and find the differential miRNA expression after the epirubicin exposure in OS cell lines. Based on the bioinfomatic research and the previous literature, we select several interesting miRNAs and validated its differential expression in OS after the epirubicin treatment by Real-time PCR. Those results imply that the differential could play important roles in the inhibitory effect of epirubicin against OS cells. Then we alter the level of the interesting miRNA in OS by the miRNA mimics transfection, so we could detect its functions and target genes in OS. We detect the OS cell proliferation, apoptosis and cell cycle distribution after the miRNA transfection alone or combined with epirubicin treatment. Moreover, we also determine the expression of its target genes which are related with the cell apoptosis and cell cycle arrest. So we could confirm the effective target genes of interesting miRNA in the anti-OS effect of epirubicin.Part I. The differential expression of miRNAs in the anti-OS progress of epirubicinObjective:To study the differential expression of miRNAs after the epirubicin exposure and validate its expression before its function investigation.Methods:We detect the inhibiroty effect of epirubicin on the OS MG-63and SAOS-2c cells by the CCK-8assay and obseve the morphologic features after the epirubicin treatment. Then we determine the miRNA expression in OS by micro-arrays and find out the differential miRNAs after epirubicin treatment. Based on the bio-inofmatics research and previous literature, we select several differential miRNAs as the interesting ones for the future function research. Finally we validate its differential expression in OS cells after the epirubicin exposure by Real-time PCR.Results:We find that1μg/ml epirubicin or more could significantly inhibit the OS cell proliferation in a dose-dependent manner. The cell morphologic features change obviously; the miRNA expressions are also dysregulatedafter1μg/ml epirubicin treatment against OS MG-63and SAOS-2cells. After the comparisons, we find out40differential miRNAs, including16over-expression ones and24down-expression. Of these, miR-302b is significantly up-regulated by the epirubicin, which might play important roles in OS cell apoptosis and cell cycle distribution.Conclusions: Epirubicin could inhibit the OS cell proliferation by the miRNA expression regulation. Forty differential miRNA might contribute to this effect and miR-302b might be one the important ones among those miRNAs.Part II. The miR-302b-mediated effect of epirubicin against osteosarcoma cells and its mechanismsObjective:To investigate the effect of miR-302b and its synergistic role with epirubicin in the anti-OS process by the overexpression of miR-302b.Methods:We validate the relative miR-302b expression in OS MG-63and SAOS-2cells after the miR-302b mimics tranfection with Lipo-2000to confirm the miR-302b restoration. After the exposure in different factors, we detect the OS cell proliferation by CCK-8; investigate the OS cell apoptosis by the Annexin V-PE/7-AAD assays and the cell cycle distribution by the PI staing. To further investigate the machanisms, we detect the Caspase-3activity and the expression levels of several apoptosis and/or cell cycle related proteins.Results:We find that miR-302b mimics transfection could increase the miR-302b expression to attenuate the OS cell proliferation and its effect could be combined with epirubicin. Over-expression of miR-302b could significantly induce OS cell apoptosis and Gl arrest to inhibit OS cell proliferation. In this process, miR-302b could activate Caspase-3, increase the Bim expression, attenuate the expression of AKT、pAKT、Bcl-2、 Cyclin D1、CDK2/4as well to induce OS cell apoptosis and G1arrest.Conclusion:Over-expression of miR-302b could regulate several key apoptosis and cell cycle related protein expression to induce OS cell apoptosis and G1arrest, inhibit cell proliferation. These mechanisms are important to mediate the anti-OS effect of epirubicin.更多还原