【作者】 潘伟波；

【导师】 叶招明；

【作者基本信息】 浙江大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 研究背景骨肉瘤是高度恶性骨肿瘤,多见于儿童和青少年,临床治疗非常困难。尽管新辅助化疗明显提高了生存率,但近30年骨肉瘤的生存率没有得到进一步的提高,5年生存率和总的生存率一直徘徊在50-60%,单纯截肢患者常于一年内死于肺转移。已经有大量的学者对骨肉瘤发生、发展的分子机制进行了深入研究,这有助于更早、更准确的诊断,以及开发新的治疗方案。尽管对骨肉瘤的分子机制的理解正在逐渐加深,但其复杂的分子机制仍然没有得到很好的了解。microRNA (miRNA,小RNA)是内源性,非编码,单链RNA,长度为19-25个核苷酸,通过促进靶mRNA降解或抑制靶mRNA水平的蛋白质翻译而在转录后水平负调控基因的表达,具有广泛的生物学效应。越来越多的资料显示miRNA参与肿瘤的发生发展,其表达谱可用于肿瘤的早期诊断,肿瘤分型并可判断肿瘤的预后以及对特定治疗方案的敏感性,甚至可以作为有效的治疗靶点。而且已经有研究发现骨肉瘤中miRNA表达谱的改变。但这些改变的miRNA表达谱对骨肉瘤的影响和意义及其内在的调控靶基因尚未进行深入的研究。因此很有必要对骨肉瘤细胞中的miRNA进行更进一步的研究。已经发现多种肿瘤中miR-27a表达升高,具有促进肿瘤的作用,而且骨肉瘤中miR-27a的表达也是升高的,但其作用机制仍不明确,本研究拟通过体外试验来明确miR-27a在骨肉瘤细胞中的作用并探讨其作用机制。研究目的1.探讨MAP2K4与miR-27a之间的靶向关系。2.构建miR-27a特异性抑制剂转染骨肉瘤细胞株MG63细胞,明确miR-27a对骨肉瘤细胞恶性生物学行为的影响。3.通过Western blotting检测JNK/p38信号通路上关键蛋白的表达水平的改变,探讨miR-27a在骨肉瘤发生发展上的分子机制。研究方法通过生物软件预测miR-27a的靶基因,构建靶基因报告载体,转染骨肉瘤细胞后进一步验证miR-27a对靶基因MAP2K4的调节作用。体外培养骨肉瘤细胞株MG63,处理组和阴性对照组分别转染miR-27a特异性抑制剂及阴性对照抑制剂,通过MTT法、集落形成试验、小室迁移试验等检测其对骨肉瘤细胞增殖、迁移和侵袭等细胞行为的影响来明确miR-27a在骨肉瘤细胞中的作用。并通过Western blotting检测JNK/p38信号通路上关键蛋白的表达水平的改变。研究结果1. MAP2K4是miR-27a的一个靶基因用三大常用的miRNA数据库TargetScan、miRBase和Pictar来预测miR-27a的目标基因。结果检测到MAP2K4mRNA的3’UTR有两个可能的结合部位。将MAP2K4的3’UTR序列克隆到pGL3载体,位于萤光素酶报告基因的下游。同时构建了预测结合部位突变的阴性对照。野生型pGL3-MAP2K43’UTR载体的萤光素酶活性明显降低,而突变型的萤光素酶活性则无明显降低。同时转染miR-27a抑制剂或阴性对照+野生型pGL3-MAP2K43’UTR载体到MG63细胞抑制miR-27a增加萤光素酶活性。2.抑制miR-27a后可以抑制MG63的增殖MTT检测发现转染72小时后,miR-27a抑制剂较阴性对照组可以显著抑制MG63细胞的增殖。MG63细胞所形成的集落较阴性对照组少了39.6%。3.抑制miR-27a后可以抑制MG63细胞的迁移和侵袭能力小室迁移和侵袭实验显示转染了miR-27a抑制剂后MG63的转移和侵袭能力分别减少了63.5%和69.1%。4.抑制MG63中miR-27a后能增加激活.JNK/p38信号通路将miR-27a抑制剂或阴性对照转染MG63细胞,结果显示转染miR-27a抑制剂的MG63细胞中MAP2K4蛋白表达水平提高的同时,JNK1和p38的磷酸化水平也分别上升了25%和29%。结论本研究发现MAP2K4是miR-27a的功能靶基因,抑制骨肉瘤细胞中的miR-27a可以提高MAP2K4的蛋白表达水平,通过激活JNK/p38信号通路抑制骨肉瘤细胞MG63的增殖、迁移和侵袭性能力。更多还原

【Abstract】 Osteosarcoma is a high-grade malignant bone neoplasm that occurs primarily in children and adolescents. The clinical treatment for osteosarcoma is very difficult. Despite incorporation of chemotherapy into initial treatment, which significantly increases the cure rate, the5-year event-free survival and overall survival rates are around50-60%, and patients treated with amputation alone often died of pulmonary metastasis within one year. Molecular pathways contributing to osteosarcoma development and progression have recently been discovered, and various studies have been carried out to investigate the genes that are involved in metastasis of osteosarcoma. However, the highly complex molecular mechanism of metastasis is still poorly understood. Recently, microRNAs have become a new research "hot topic" for molecular pathways. MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs of19-25nucleotides in length, which can regulate gene expression at the post-transcriptional level by inhibiting the translation of a protein at the mRNA level or by promoting mRNA degradation. However, their biological function remains largely unknown and only a few mRNAs that are directly regulated by miRNAs in animals have been verified empirically. Comparison between human cancer and their normal tissue counterparts have revealed distinct miRNAs expression profiles. In addition, miRNAs may function as either oncogenes or tumor suppressors by specifically regulating the expression of their target genes. miR-27a, a member of an evolutionarily conserved miRNA family, is abnormally increased in several types of cancers and has been identified as being increased in osteosarcoma and has a pro-metastatic role in osteosarcoma cell lines. However, the effects of miR-27a on osteosarcoma have not been totally elucidated. Therefore, it is of great significance to further study the mechanism of miR-27a in osteosarcoma.Methods:In this study, Three Biological softwares were conducted to predict miR-27a target in MAP2K4. a specific miR-27a inhibitor was used to inhibit endogenous miR-27a activity in human osteosarcoma cell line MG63. Cell proliferation assay, colony formation assay, migration and invasion assay were performed to assess the effect of miR-27a on proliferation, metastasis and invasion of MG63. The expression levels of several proteins evolved in the JNK/p38signaling passage was detected by Western blotting.Results:The luciferase activity of the wild-type pGL3-MAP2K43’UTR vector was significantly inhibited after the transfection of miR-27a precursor or the control precursor into MG63cells, but not by the mutant-type pGL3-MAP2K43’UTR vector. Meanwhile, inhibition of miR-27a increased the luciferase activity after the transfection of miR-27a inhibitor or the control inhibitor into MG63cells with the wild-type pGL3-MAP2K43’UTR vector. These results demonstrated that MAP2K4is a potential target of miR-27a and can be directly regulated by miR-27a. Inhibition of miR-27a significantly suppressed cell proliferation after72hours compared with the negative control group, and statistical analysis indicated that inhibition of miR-27a suppressed the colony formation of MG63cells by39.6%. Transwell migration and invasion assays demonstrated that the number of migratory and invasive cells transfected with the miR-27a inhibitor was reduced by63.5%and69.1%, respectively. After transfection with miR-27a inhibitor in MG63cells, the level of phosphor-JNK1and phosphor-p38raised (by25%and29%, respectively) along with the up-regulation of MAP2K4protein.In summary, this is the first study to propose that miR-27a functions as an oncogene by targeting MAP2K4in osteosarcoma MG63cell lines. Inhibition of miR-27a increases MAP2K4, which in turn inhibits cell proliferation and migration through the JNK/p38signaling pathway in MG63cells. These findings may help us to understand the molecular mechanism of miR-27a in tumorigenesis of osteosarcoma and provide new diagnostic and therapeutic options for treatment of this neoplasia.更多还原