【作者】 闫康；

【导师】 马保安； 杨彤涛；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 硕士

【摘要】 miRNAs(microRNAs)是一类内源性、非编码蛋白的单链小分子RNA。其大小约为20~25nt,编码基因约占整个基因组的1%。miRNA参与基因的转录后调节,对基因表达具有重要调控作用。研究表明miRNA在各种真核细胞中广泛存在,参与生物体的生长发育、信号转导、组织分化、疾病发生等过程,还决定其它很多生物体发育和行为的变化。同时, miRNA在多种肿瘤的发生、发展中也扮演着相当重要的角色。miR-34a最早在C. elegans(Caenorhabditis elegan,秀丽隐杆线虫)中被发现,是一个进化保守miRNA家族miR-34s中的一员。Welch等最早将miR-34a与肿瘤发生发展联系起来。他们发现miR-34a的编码基因位于染色体1p36,并常在人神经母细胞瘤中缺失。miR-34a受经典抑癌基因p53的直接调控,在miR-34a上游约30kb区域部位有p53高度保守的结合位点。miR-34a通过下调CDK4、CDK6、E2F3、E2F5、c-Met等癌基因发挥其肿瘤抑制作用。在p53引起细胞周期的G1期阻滞、细胞衰老、凋亡等生物学行为中,miR-34a同样扮演着重要的角色。miR-34a的失活或缺失可能与多种肿瘤的发病机理有关,但目前miR-34a在骨肉瘤中的功能鲜有报道。因此,研究miR-34a在骨肉瘤中的功能、作用机制并进一步探索其临床应用具有重要意义。在前期的研究中,我们通过microRNA基因芯片技术对不同转移能力骨肉瘤细胞系(E10、H9)中差异表达的miRNA进行分析发现,miR-34a在E10细胞中的表达下调,所以本实验将在此基础上对miR-34a与骨肉瘤之间的关系进行更进一步的研究。研究目的:验证在不同转移能力骨肉瘤细胞系(E10、H9)中miR-34a的差异表达;研究miR-34a的在骨肉瘤SOSP-9607细胞中的生物学功能并进一步研究其作用机制,为miR-34a在骨肉瘤预后判断和治疗等方面的临床应用提供理论基础。研究方法:1.采用Realtime RT-PCR和Northern blotting分析的方法,对前期用miRNA基因芯片筛选出来的在不同转移能力骨肉瘤细胞系(E10、H9)中差异表达的miR-34a基因进行验证。2.构建人miR-34a的真核表达载体,用其转染SOSP-9607细胞,并用G418筛选miR-34a稳定表达细胞系,为进一步研究miR-34a在骨肉瘤中的功能及基因调控机制奠定实验基础。3.用pcDNA-miR34a或pcDNA3.1(+)转染骨肉瘤SOSP-9607细胞,观察其在骨肉瘤SOSP-9607细胞增殖,侵袭和迁移等方面的作用。构建裸鼠的骨肉瘤原位成瘤肺转移模型,并进一步研究miR-34a对骨肉瘤细胞原位成瘤能力和肺转移能力的影响。4.通过miRanda,TargetScan和PicTar等生物信息学计算方法,分析miR-34a可能的靶基因,并结合靶基因的功能最终确定待验证的靶基因。研究结果:1.分别用Northern blotting和Realtime RT-PCR的方法对miR-34a在E10、H9细胞中的表达进行验证,结果显示miR-34a在E10中的表达明显降低,与芯片检测的结果一致。2.成功构建了人miR-34a的真核表达载体pcDNA-miR34a。pcDNA-miR34a可于骨肉瘤SOSP-9607细胞中上调miR-34a的表达。获得了miR-34a高表达骨肉瘤细胞系。3.在体外,外源性miR-34a对SOSP-9607细胞的增殖,侵袭和迁移能力有明显的抑制作用。动物实验中,miR-34a对骨肉瘤的原位成瘤能力和肺转移能力同样有显著的抑制作用。4.通过miRanda、TargetScan和PicTar等生物信息学计算方法,分析miR-34a可能的靶基因,并结合靶基因的功能最终选出了几个待验证的miR-34a功能相关靶基因。分别为:c-Met、Bcl2、Notch -1和Notch-2。结论:1. miR-34a在不同转移能力骨肉瘤细胞系中差异表达,与骨肉瘤转移具有相关性。有必要对miR-34a在骨肉瘤转移中的功能及基因调控机制作进一步的深入研究。2.成功构建了人miR-34a的真核表达载体pcDNA-miR34a,并在骨肉瘤细胞SOSP-9607细胞中有效表达,为进一步研究miR-34a在骨肉瘤中的功能及基因调控机制奠定了实验基础。3. miR-34a具有显著的抑癌生物活性,将有可能成为骨肉瘤治疗的新靶点。4.通过生物信息学方法,初步确定了与miR-34a功能有可能相关的靶基因,为进一步验证miR-34a的靶基因指明了方向、奠定了基础。更多还原

【Abstract】 miRNA (microRNA) is a class of endogenous non-protein-coding single-stranded small RNA, which is about 20-25 nt in length. Its coding genes account for approximately 1% of the entire genome. miRNA plays an important role in the post-transcriptional regulation of gene expression. Studies showed that miRNA expression is prevalent in a variety of eukaryotic cells and involved in the regulation of growth and development of organisms, signal transduction, tissue differentiation, disease occurrence and so on. Meanwhile, studies also showed that miRNA plays an important role in the occurrence and development of a variety of tumors.miR-34a, which was initially found in C. elegans (Caenorhabditis elegans), is a member of an evolutionarily conserved miRNA family, miR-34s. miR-34a, whose encoding gene is on chromosome 1p36, is correlated with tumor occurrence and frequently missed in human neuroblastoma. miR-34a is directly regulated by p53 tumor suppressor. There is a highly conserved p53 binding site which is approximately 30kb above the miR-34a encoding gene. miR-34a plays the tumor inhibitory role by down-regulating its targets such as CDK4, CDK6, E2F3, E2F5, c-Met and so on. miR-34a also plays an important role in the p53-induced G1 phase arrest of cell cycle, cell senescence, apoptosis and other biological behavior. The inactivation and absence of miR-34a may be related to the pathogenesis of a variety of tumors. However, the relationship between osteosarcoma and miR-34a has been reported rarely. Therefore, it is of great significance to study the fuction and mechanism and to further explore the clinical application of miR-34a in osteosarcoma.Previously, we have evaluated the expression of miR-34a in osteosarcoma with different metastatic capacities by using microRNA microarray assay. The results show that the expression of miR-34a was down-regulated in high-metastatic osteosarcoma cell lines E10. Therefore, in this study, we will further study the function and mechanism of miR-34a in osteosarcoma.Objective:To verify the differential expression of miR-34a in the osteosarcoma cell lines with different metastatic capacities (E10, H9); To study the function and mechanism of miR-34a in osteosarcoma SOSP-9607 cells in order to further promote the clinical application of miR-34a in osteosarcoma prognosis and treatment.Methods:1. To verify the differential expression of miR-34a in the osteosarcoma cell lines with different metastatic capacities (E10, H9) by using Northern blotting and Realtime RT-PCR.2. To construct a recombinant eukaryotic expression plasmid of miR-34a, pcDNA-miR34a. To transfect it into osteosarcoma SOSP-9607 cells, and obtain G418-selected stable cell lines. 3. To evaluate the effects of miR-34a on proliferation, invasion and migration of osteosarcoma in vitro. To construct an osteosarcoma in situ tumor formation and pulmonary metastasis model, and then evaluate the effects of miR-34a on the capacities of in situ tumor formation and pulmonary metastasis.4. To predict miR-34a target genes by using bioinformatics calculation methods, such as miRanda, TargetScan and PicTar in combining with the fuctions of target genes. Ultimately, to selected the potential target genes that will be further verified.Results:1. We evaluated the differential expression of miR-34a in the osteosarcoma cell lines with different Metastatic capacities (E10, H9) by using Northern blotting and Realtime RT-PCR. The results showed that miR-34a was lower expressed in E10 cells as compared with H9 cells, which were consistent with the previous results from microRNA microarray assay.2. The human miR-34a eukaryotic expression vector pcDNA-miR34a was successfully constructed. pcDNA-miR34a can up-regulate the expression of miR-34a in osteosarcoma SOSP-9607 cells. Finally, we obtained stable osteosarcoma cells which expressed a high level of miR-34a.3. In vitro, miR-34a significantly inhibits the proliferation, invasion and migration of SOSP-9607 cells. And in vivo, miR-34a also remarkably inhibits the capacities of in situ tumor formation and pulmonary metastasis.4. We predicted miR-34a target genes by using bioinformatics methods, such as miRanda, TargetScan and PicTar in combining with the fuctions of target genes. Ultimately, we selected potential target genes that would be further verify. They are c-Met, Bcl2, Notch1 and Notch2.Conclusion: 1. The expression of miR-34a in osteosarcoma cells, which is different in E10 and H9 cells, is closely correlated with the capacity of metastasis. Thus, it is of great significance to to further study the fuction and mechanism of miR-34a in osteosarcoma.2. The eukaryotic expression plasmid of miR-34a was successfully constructed and effectively expressed in osteosarcoma SOSP-9607 cells. This facilitated the further investigations of function and mechanism of miR-34a in osteosarcoma.3. Functioning as a tumor suppressor, miR-34a can be used as a potential target for the treatment of osteosarcoma.4. We predicted miR-34a target genes by using bioinformatics methods, and initially chosed three target genes that may correlate to the fuction of miR-34a. This facilitated the further verification of miR-34a target genes.更多还原