circ-GRB10-miR-328-5p-ERBB2通路调控髓核细胞凋亡的实验研究

发布时间：2018-04-27 04:14

本文选题：椎间盘退行性改变 + 环状RNA　；参考：《天津医科大学》2017年硕士论文

【摘要】：[目的]腰背痛已经成为全世界范围内最常见的肌肉骨骼疾病之一,具有发病率高、治疗难度大、远期治疗效果欠佳的特点。研究显示许多因素都可以导致腰背部疼痛,椎间盘退行性改变(intervertebral disk degeneration,IDD)被认为是其中最重要的因素之一。IDD是一个由细胞介导的异常反应,包括:1.髓核(nucleus pulposus,NP)部分基质成分合成减少;2.基质金属蛋白酶含量的增加;3.NP细胞的凋亡及死亡。研究表明非编码RNA (non-coding RNA,ncRNA)广泛存在于生命体当中,而且可以通过调节基因的表达在许多生物学过程中发挥重要的作用。环状RNA (cicular RNA,circRNA)是近来新发现的一类ncRNA,可通过吸附微小RNA (microRNA,miRNA)在细胞质中作为miRNA海绵,发挥竞争性内源RNA(competitive endogenous RNA,ceRNA)的作用,起到促进miRNA靶基因表达的作用。现已证实RNA调控网络的异常在多种疾病的发生、发展过程中都起到重要作用。然而,目前circRNA在IDD过程中的调控作用及相关机制尚缺乏报道。本实验旨在利用生物信息学方法寻找IDD过程中起关键调控作用的circRNA,并探索其在IDD过程中的调控机制。[方法]1.通过HE染色方法观察退变的NP组织的形态学改变。2.通过TUNEL荧光染色观察退变的NP组织中NP细胞的凋亡情况。3.从GEO数据库下载的正常NP组织和退变的NP组织中circRNA和microRNA芯片数据并进行差异表达分析,寻找关键circRNA以及与其表达呈负性相关的microRNA。4.通过荧光原位杂交的方法检测关键circRNA和与其存在显著负相关的microRNA在NP细胞内定位。5.采用荧光素酶报告基因实验检测关键circRNA和microRNA的结合位点。6.随后利用 miRPathDBv1.O、DIANA-miRPathv3.0、TargetScan数据库预测关键microRNA调控的通路,同时利用DIANA,miRTarBase,TargetScan,miRDB数据库预测该microRNA靶基因。7.在NP细胞内分别过表达关键circRNA和关键microRNA,利用Western blotting和RT-PCR检测靶基因的表达情况。8.通过流式细胞仪检测关键circRNA对NP细胞凋亡的影响。[结果]本研究通过检索GEO数据库中关于IDD的circRNA芯片数据,构建circRNA在退变的椎间盘NP细胞中的表达谱,随后通过生物信息学方法筛选出104条差异表达的circRNA,并且预测出343个microRNA应答元件。近一步结合样本来源相同的miRNA芯片数据及相关性分析,预测出circ-GRB1O和miR-328-5p的相互调控关系。通过原位杂交实验验证了 circ-GRB10和miR-328-5p在NP细胞质中存在共定位关系,并且观察到circ-GRB10和miR-328-5p在NP细胞中表达量的变化,近一步证实了芯片分析的结果。在circ-GRB1O的生物功能学验证的实验部分,本研究通过荧光素酶报告基因实验证实了 circ-GRB10作为miR-328-5p海绵的作用,并且确定了 circ-GRB10和 miR-328-5p 的相互作用位点。通过 miRPathDBv1.O、DIANA-miRPathv3.0、TargetScan数据库预测miR-328-5p的调控通路,同时利用DIANA,miRTarBase,TargetScan,miRDB数据库预测miR-328-5p靶基因,并与上述通路信息相结合,本研究最后确定ERBB2为miR-328-5p的靶基因。随后在细胞水平上,本研究证实:1. NP细胞中过表达miR-328-5p可以下调ERBB2的表达,而circ-GRB1O可以抑制miR-328-5p作用,上调NP细胞中ERBB2的含量;2.在NP细胞中抑制circ-GRB1O的表达,可以下调ERBB2的含量,并且促进NP细胞的凋亡。[结论]1.本研究表明大量circRNA参与了 IDD的发生、发展过程,并且circ-GRB 10作为IDD过程中的关键circRNA具有抑制NP细胞凋亡的重要作用。2.揭示了 circ-GRB10-miR-328-5p-ERBB2调控网络的平衡在NP细胞凋亡过程中的重要作用。3.为IDD的发生、发展过程中相关机制的研究提供了新的思路。
[Abstract]:[Objective] low back pain has become one of the most common musculoskeletal diseases worldwide, characterized by high incidence, difficulty in treatment, and poor long-term treatment. Research shows that many factors can lead to pain in the back and intervertebral disk degeneration (IDD) is considered to be the most important. One of the factors,.IDD, is a cell mediated abnormal reaction, including a decrease in the composition of part of the 1. nucleus (nucleus pulposus, NP), the increase in the content of the 2. matrix metalloproteinase, the apoptosis and death of the 3.NP cells. The study shows that the non coded RNA (non-coding RNA, ncRNA) exists widely in the life body and can be used by the regulatory group. The expression of the cause plays an important role in many biological processes. Cicular RNA (circRNA) is a newly discovered class of ncRNA, which can play the role of competitive endogenous RNA (competitive endogenous) by adsorbing small RNA (microRNA, miRNA) as a miRNA sponge in the cytoplasm, to promote the expression of the target gene expression. It has been proved that the abnormality of the RNA regulatory network plays an important role in the development and development of various diseases. However, the regulatory role and related mechanisms of circRNA in the IDD process are not yet reported. This experiment aims to explore the key regulatory role of circRNA in the process of IDD using bioinformatics methods and explore its IDD in the process. Regulatory mechanism in the process. [method]1. observe the morphological changes of the degenerative NP tissue by HE staining method.2. through TUNEL fluorescence staining to observe the apoptosis of NP cells in the NP tissue of the degenerated.3.,.3. from the normal NP tissue and the NP tissue from the GEO database and the differential expression analysis of the circRNA and chip data in the NP tissue of the degenerative NP. Key circRNA and microRNA.4., which are negatively related to its expression, detect key circRNA and its significant negative correlation microRNA in NP cells by fluorescence in situ hybridization..5. uses luciferase reporter gene test to detect the binding site of key circRNA and microRNA, and then uses miRPathDBv1.O, DIANA-miRPat Hv3.0, TargetScan database predicts critical microRNA regulation pathway, and uses DIANA, miRTarBase, TargetScan, miRDB database to predict the key circRNA and key microRNA in NP cells. The effect of key circRNA on the apoptosis of NP cells. [results] by searching the circRNA chip data of IDD in the GEO database, the expression of circRNA in the degeneration of intervertebral disc NP cells was constructed, and then 104 differentially expressed circRNA were screened by bioinformatics method, and 343 microRNA response elements were predicted. The same miRNA chip data and correlation analysis were used to predict the mutual regulation relationship between circ-GRB1O and miR-328-5p. The co localization relationship between circ-GRB10 and miR-328-5p in the cytoplasm of NP was verified by in situ hybridization, and the changes in the expression of circ-GRB10 and miR-328-5p in NP cells were observed, and the core was confirmed in the last step. The results of the slice analysis. In the experimental part of circ-GRB1O's biofunctional verification, this study confirmed the role of circ-GRB10 as a miR-328-5p sponge by luciferase reporter gene experiment, and determined the interaction sites between circ-GRB10 and miR-328-5p. The prediction of miR by miRPathDBv1.O, DIANA-miRPathv3.0, TargetScan database. -328-5p regulation pathway, simultaneously using DIANA, miRTarBase, TargetScan, miRDB database to predict miR-328-5p target gene, and combined with the pathway information, this study finally determined the ERBB2 as the target gene for miR-328-5p. Then at the cell level, this study confirmed that 1. NP fine cell overexpressed miR-328-5p can downregulate ERBB2 expression, and circ-G RB1O can inhibit the effect of miR-328-5p and increase the content of ERBB2 in NP cells; 2. inhibiting the expression of circ-GRB1O in NP cells can reduce the content of ERBB2 and promote the apoptosis of NP cells. [conclusion]1. this study showed that a large number of circRNA participated in IDD occurrence, development process, and circ-GRB 10 was a key inhibitor in the process. The important role of NP cell apoptosis.2. reveals that the balance of circ-GRB10-miR-328-5p-ERBB2 regulatory network plays an important role in the process of NP cell apoptosis,.3. is the occurrence of IDD, and the research of related mechanisms in the process of development provides a new way of thinking.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.55

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