miR-10a靶向调控Bcl-6对弥漫大B细胞淋巴瘤生物学功能的影响

发布时间：2018-07-10 08:07

本文选题：非霍奇金淋巴瘤 + 弥漫大B细胞淋巴瘤　；参考：《天津医科大学》2017年博士论文

【摘要】：研究背景:随着人类基因组计划的完成及高通量基因测序和转录组学研究的不断深入,研究人员发现基因的精准表达调控对维持机体稳态和正常的生理活动至关重要。非编码RNA (non-coding RNA,ncRNA)是不存在编码蛋白能力RNA的统称,可通过多种机制在RNA水平干预基因调控网络,并广泛参与细胞代谢、分化、增殖等一系列生命进程。微小RNA (microRNA, miRNAs)作为非编码RNA重要的一员,主要通过与靶基因mRNA配对,沉默靶基因表达发挥作用。通常认为,一种miRNA可以通过识别靶基因的mRNA3'UTR区域的互补结合序列,调控多个靶基因活性、稳定性,进而影响相应靶基因干扰细胞功能;多种miRNAs也可以识别干扰同一个靶基因对其下游信号通路产生网络交互调控影响;因此,miRNAs表达异常可经由复杂的基因交互网络参与细胞表观遗传调控、细胞自噬调控、糖代谢调控以及肿瘤相关基因调控等发挥强大的生物学功能,是生命科学领域专家关注的热点。由于50%以上的miRNAs基因位于肿瘤相关基因组区域/脆性位点,因此在人类肿瘤发生过程中起到重要作用,而miRNAs在肿瘤中的重要意义促使“肿瘤相关miRNAs”这一概念的提出,即“Oncomirs”。为此,寻找疾病特异性miRNAs并明确其调控机制,对疾病诊断、治疗、预后均有重要意义。弥漫大B细胞淋巴瘤(Diffuse large B cell lymphoma, DLBCL)是侵袭性非霍奇金淋巴瘤(non-Hodgkin lymphoma NHL)中最常见的亚型,我国弥漫大B细胞淋巴瘤的发病率约为非霍奇金淋巴瘤的30%-45%。近十余年,随着以美罗华为主的免疫化疗方案(R-CHOP)广泛应用于弥漫大B细胞淋巴瘤的治疗,半数以上的患者获得治愈,但仍有超过30%的患者面临复发的风险,复发难治病例通常对现有药物敏感度差,亟需新型治疗措施。miRNAs替代治疗是目前生命科学领域的一个热点,大量研究从事疾病差异表达miRNAs的确定工作,并对其发挥作用的相应靶基因进行验证,为寻找肿瘤可药性miRNAs提供理论基础。miR-10a是一个重要的肿瘤相关性miRNA,已有研究证实miR-10a在脑胶质瘤、卵巢癌、以及非霍奇金淋巴瘤中存在表达差异,并可通过靶基因调控、免疫调控等多种机制发挥抑癌作用,但miR-10a在弥漫大B细胞淋巴瘤中的研究未见报道。本课题组前期研究发现弥漫大B细胞淋巴瘤患者血浆中miR-10a表达较健康对照组显著降低,因此我们猜测miR-10a在弥漫大B细胞淋巴瘤的恶性转化过程中发挥作用,并拟研究其对弥漫大B细胞淋巴瘤生物学功能的影响。研究目的:本研究旨在探讨miR-10a在弥漫大B细胞淋巴瘤中表达情况及其在弥漫大B细胞淋巴瘤发生发展过程中的生物学功能,寻找miR-10a下游作用靶基因,并进一步探讨这种靶向作用对于弥漫大B细胞淋巴瘤细胞生物学功能的影响的作用机制,以期为弥漫大B细胞淋巴瘤的靶向治疗提供新的思路及途径。研究方法:1.收集弥漫大B细胞淋巴瘤组织及作为对照的非肿瘤性淋巴组织,使用qRT-PCR评估miR-10a在弥漫大B细胞淋巴瘤组织及对照组织中的表达水平。2.使用miR-10a过表达慢病毒质粒构建miR-10a差异过表达细胞系,通过移植瘤模型验证miR-10a对弥漫大B细胞淋巴瘤肿瘤生成的影响。使用细胞转染技术干预细胞内miR-10a表达水平,经qRT-PCR检验miR-10a转染效率,通过流式细胞术、细胞周期实验、CCK8细胞增殖实验在细胞水平验证miR-10a对弥漫大B细胞淋巴瘤细胞生物学功能的影响。3.利用生物信息学软件TargetScan、miRanda、PicTar并结合文献检索,预测miR-10a潜在靶基因,使用双荧光素酶报告基因的方法进行验证;通过细胞转染技术干预细胞内miR-10a及相应靶基因表达,使用Western blot技术验证miR-10a对其靶基因的表达水平的调控作用;借助流式细胞术、细胞周期实验、CCK8细胞增殖实验在细胞水平验证miR-10a确通过调控其下游靶基因的表达水平,影响弥漫大B细胞淋巴瘤细胞生物学功能。研究结果:1.相较于对照的非肿瘤性淋巴瘤组织,miR-10a在弥漫大B细胞淋巴瘤肿瘤组织中表达水平明显降低。2.动物移植瘤模型示miR-10a可在动物水平抑制弥漫大B细胞淋巴瘤生成;细胞层面实验显示:过表达miR-10a可以促进弥漫大B细胞淋巴瘤细胞凋亡、抑制细胞增殖;而弥漫大B细胞淋巴瘤miR-10a表达水平受到抑制时,细胞凋亡率降低、细胞增殖活性增强。3.生物信息学预测提示Bcl-6为miR-10a潜在调控靶点;双荧光素酶实验验证Bcl-6和miR-10a存在靶向调控关系。Bcl-6在弥漫大B细胞淋巴瘤组织中的表达水平较对照正常组织明显升高。过表达弥漫大B细胞淋巴瘤细胞内miR-10a水平,Bcl-6的表达受抑制;抑制细胞内miR-10a表达水平,Bcl-6的表达水平升高。Bcl-6能够抑制细胞凋亡、促进细胞增殖,且miR-10a和Bcl-6对弥漫大B细胞淋巴瘤的生物学功能成负调控模式。研究结论:1.弥漫大B细胞淋巴瘤组织中miR-10a表达水平较反应增生性组织明显降低。2. miR-10a促进弥漫大B细胞淋巴瘤细胞凋亡,并抑制其增殖。3.在弥漫大B细胞淋巴瘤细胞中,miR-10a通过靶向调控Bcl-6促进细胞凋亡、抑制细胞增殖。
[Abstract]:Background: with the completion of the human genome project and the continuous deepening of high throughput gene sequencing and transcriptional studies, researchers have found that the precise regulation of gene expression is essential to maintain the homeostasis and normal physiological activities of the body. Non coded RNA (non-coding RNA, ncRNA) is a general name for the absence of the ability to code protein, RNA. Many mechanisms interfere with the gene regulation network at RNA level, and participate in a series of life processes, such as cell metabolism, differentiation and proliferation. As an important member of non coded RNA, micro RNA (microRNA, miRNAs) is mainly matched with the target gene mRNA to silence the target gene expression and volatiles. It is generally believed that a miRNA can be identified by the target group. The complementary binding sequence of the mRNA3'UTR region regulates the activity and stability of multiple target genes, and then affects the function of the target genes to interfere with the cell function, and a variety of miRNAs can also identify the interference of the same target gene to the downstream signaling pathway of the target gene. Therefore, the abnormal expression of miRNAs can be mediated by a complex gene interaction network. Cell epigenetic regulation, regulation of autophagy, regulation of cell autophagy, regulation of glucose metabolism, and regulation of tumor related genes play a very important role in the field of life science. More than 50% of miRNAs gene is located in the region of the tumor related genome / fragile site, so it plays an important role in the process of human cancer. The significance of miRNAs in the tumor promotes the concept of "tumor related miRNAs", namely, "Oncomirs". For this purpose, it is important to find the disease specific miRNAs and clarify its regulatory mechanism for the diagnosis, treatment, and prognosis of the disease. Diffuse large B cell lymphoma (Diffuse large B cell lymphoma, DLBCL) is an invasive non Hodgkin. The most common subtype of non-Hodgkin lymphoma NHL, the incidence of diffuse large B cell lymphoma in our country is about ten years in non Hodgkin's lymphoma, and with the immunotherapy (R-CHOP) based on the most widely used for the treatment of diffuse large B cell lymphoma, more than half of the patients have been cured. More than 30% of the patients still face the risk of relapse. The relapsed and refractory cases are usually poor in sensitivity to existing drugs. It is urgent for the new treatment measures to replace.MiRNAs as a hot spot in the field of life science. MiRNAs provides a theoretical basis for finding tumor drug-resistant miRNAs..miR-10a is an important tumor associated miRNA. There has been a study on the expression of miR-10a in glioma, ovarian cancer, and non Hodgkin's lymphoma, and can play a role in inhibiting cancer by targeting gene regulation, immunoregulation and so on, but miR-10a is diffuse to large B cells. There is no report in the study of Ba tumor. Our previous study found that the expression of miR-10a in the plasma of diffuse large B cell lymphoma was significantly lower than that of the healthy control group. Therefore, we speculate that miR-10a plays a role in the malignant transformation of diffuse large B cell lymphoma and is intended to study the biological function of diffuse large B cell lymphoma. The purpose of this study is to investigate the expression of miR-10a in diffuse large B cell lymphoma and its biological functions in the development of diffuse large B cell lymphoma, to find the target gene for the downstream effect of miR-10a, and to further explore the effect of this target on the biological function of diffuse large B cell lymphoma cells. The mechanism of action is expected to provide new ideas and ways for the targeting therapy of diffuse large B cell lymphoma. Research methods: 1. collect diffuse large B cell lymphoma and non tumor lymphoid tissue as control, and use qRT-PCR to evaluate the expression level of miR-10a in diffuse large B cell lymphoma tissue and control tissues using miR-10a over miR-10a The expression of lentivirus plasmid was expressed by miR-10a differentially overexpressed cell lines, and the effect of miR-10a on the formation of diffuse large B cell lymphoma was verified by transplantation tumor model. Cell transfection technique was used to intervene the level of miR-10a expression in cell, qRT-PCR test of miR-10a transfection efficiency by flow cytometry, cell cycle experiment, and CCK8 cell proliferation. The effect of miR-10a on the biological function of diffuse large B cell lymphoma (.3.) was tested at the cell level. The bioinformatics software TargetScan, miRanda, PicTar and literature retrieval were used to predict the potential target genes of miR-10a, and the method of using the double luciferase reporter gene was tested, and the cell transfection technique was used to interfere in the intracellular miR-10a and the cell transfection technique. The expression of target gene and Western blot technique were used to verify the regulation of miR-10a on the expression level of the target gene. By flow cytometry, cell cycle experiment, and CCK8 cell proliferation test, the expression level of the target gene was regulated by miR-10a, and the biological function of diffuse large B cell lymphoma cells was influenced by miR-10a. Results: 1. compared with control non tumor lymphoma tissue, the expression level of miR-10a in diffuse large B cell lymphoma tumor tissue decreased significantly in.2. animal transplantation tumor model and miR-10a could inhibit the formation of diffuse large B cell lymphoma in animal level; the cell level experiment showed that overexpression of miR-10a could promote diffuse large B cell lymphomas Apoptosis and inhibition of cell proliferation, while the miR-10a expression level of the diffuse large B cell lymphoma was inhibited, the apoptosis rate was reduced, the proliferation activity enhanced by.3. bioinformatics prediction suggested that Bcl-6 was a potential target for miR-10a, and the dual luciferase experiment proved that Bcl-6 and miR-10a had a targeting regulation relationship with.Bcl-6 in the diffuse large B thin The expression level of cell lymphoma was significantly higher than that of normal control. Overexpression of miR-10a level in large B cell lymphoma cells was inhibited, the expression of Bcl-6 was inhibited, the expression level of miR-10a in cells was inhibited, and.Bcl-6 could inhibit apoptosis and promote cell proliferation by the expression level of Bcl-6, and miR-10a and Bcl-6 were diffuse large B cells. The biological function of lymphoma is negatively regulated. Conclusion: 1. the expression level of miR-10a in 1. diffuse large B cell lymphoma significantly reduces the apoptosis of diffuse large B cell lymphoma cells and inhibits the proliferation of.3. in diffuse large B cell lymphoid cells, and miR-10a promotes Bcl-6 in the diffuse large B cell lymphomas. Apoptosis and inhibition of cell proliferation.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.1

【参考文献】