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Defining the post-transcriptional regulatory mechanisms controlling the SOX9 gene and their potential for promoting cartilage regeneration

定义控制 SOX9 基因的转录后调控机制及其促进软骨再生的潜力

基本信息

批准号：
BB/K00381X/1
负责人：
Simon Tew
金额：
$ 43.05万
依托单位：
University of Liverpool
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FK00381X%2F1
关键词：
Defining post transcriptional regulatory mechanisms

项目摘要

Articular cartilage is a tissue in our joints that allows easy articulation and provides "shock absorbing" properties. Following injury or disease it heals very poorly. The tissue is typically described as having one type of cell in it, called a chondrocyte and it is very important that this cell type retains its ability to effectively produce the correct type of connective tissue and allow cartilage to perform its function properly. A very important gene, called SOX9, is used by chondrocytes to orchestrate the correct production of cartilage tissue and it is absolutely essential to the formation of healthy cartilage. Regulation of SOX9 has been implicated in age-related diseases such as osteoarthritis and its use in gene therapy approaches is able to promote the engineering of cartilage tissue. The ability to promote the activity of the SOX9 gene in chondrocytes is thus desirable but its regulation by these cells is still not fully understood. The applicant has been able to demonstrate that a regulatory process that controls the decay of an important gene intermediate, called mRNA, can affect the activity of the SOX9 gene. This project takes the work forward by examining the molecular mechanisms behind this process and use the findings to improve the quality of engineered cartilage.This project will firstly build upon preliminary data, which shows that a group of specialised regulatory proteins, which can interact with mRNA and alter its decay rate, inhibit the activity of the SOX9 gene. Using a cell culture system, these proteins will be inhibited and the effect this has on SOX9 gene function will be measured. The effect of inhibiting these proteins will then be combined with the manipulation of other DNA-like molecules called microRNAs that exist in chondrocytes. Overall, the first objective will define combination of proteins and microRNAs, which potently affect the activity of the SOX9 gene. The second objective of the project is to determine what parts of the SOX9 gene make it a target for mRNA decay. By introducing a molecular "reporter" into chondrocytes, the contribution of different regions of the gene to this decay will be assessed. This work will seek to determine whether certain regulatory parts of the SOX9 gene are responsible for the general instability of the mRNA whilst others allow changes to be made to the rate of decay in response to environmental cues. Further experiments will then be conducted to examine how these parts of the SOX9 gene interact with the specialised mRNA interacting proteins examined during the first objective. The first two objectives of the project examine the basic biological mechanisms, which control an important aspect of SOX9 gene regulation. These are valuable experiments, which will provide an understanding of the control of an essential cartilage gene and of how its altered activity may contribute to the development of musculoskeletal diseases in the aged. The final aim of the project is to take these findings and use them to increase SOX9 gene activity, by reducing mRNA decay through the manipulation of relevant mRNA interacting proteins and microRNAs in human adult stem cells. The effect of these interventions on the quality and quantity of cartilage tissue produced by the stem cells will then be assessed.To conclude, the outcomes of the project will be the identification of factors which regulate the SOX9 gene, the definition of the characteristics of the gene that make it susceptible to this regulation and the evaluation of the potential of these findings to improve our ability to engineer cartilage tissue. In delivering these outcomes the project aims to better understand the fundamental regulation of a critical cartilage gene, which will impact on how we understand age-related tissue pathology and improve tissue regeneration techniques.

关节软骨是我们关节中的一种组织，可以轻松连接并提供“减震”特性。在受伤或生病后，它愈合得很差。该组织通常被描述为具有一种类型的细胞，称为软骨细胞，并且这种细胞类型保持其有效产生正确类型的结缔组织的能力并允许软骨正确地执行其功能是非常重要的。一个非常重要的基因，称为SOX 9，被软骨细胞用来协调软骨组织的正确生产，它对健康软骨的形成至关重要。SOX 9的调节与骨关节炎等年龄相关疾病有关，其在基因治疗方法中的应用能够促进软骨组织的工程化。因此，促进软骨细胞中SOX 9基因活性的能力是期望的，但这些细胞对其的调节仍然没有完全理解。申请人已经能够证明，控制重要基因中间体（称为mRNA）衰变的调控过程可以影响SOX 9基因的活性。该项目将通过研究这一过程背后的分子机制来推进这项工作，并利用这些发现来改善工程软骨的质量。该项目将首先建立在初步数据的基础上，这些数据表明一组专门的调节蛋白可以与mRNA相互作用并改变其衰减速率，抑制SOX 9基因的活性。使用细胞培养系统，这些蛋白质将被抑制，并将测量其对SOX 9基因功能的影响。然后，抑制这些蛋白质的作用将与操纵软骨细胞中存在的称为microRNA的其他DNA样分子相结合。总的来说，第一个目标将定义蛋白质和microRNA的组合，这可能会影响SOX 9基因的活性。该项目的第二个目标是确定SOX 9基因的哪些部分使其成为mRNA衰变的靶点。通过将分子“报告”引入软骨细胞，将评估基因的不同区域对这种衰变的贡献。这项工作将试图确定SOX 9基因的某些调控部分是否负责mRNA的一般不稳定性，而其他部分则允许响应环境线索而改变衰变速率。然后将进行进一步的实验，以检查SOX 9基因的这些部分如何与第一个目标期间检查的专门mRNA相互作用蛋白相互作用。该项目的前两个目标是研究控制SOX 9基因调控的一个重要方面的基本生物学机制。这些都是有价值的实验，这将提供一个基本的软骨基因的控制和它的活动如何改变可能有助于在老年人的肌肉骨骼疾病的发展的理解。该项目的最终目的是利用这些发现并利用它们来增加SOX 9基因的活性，通过操纵人类成体干细胞中相关的mRNA相互作用蛋白和microRNA来减少mRNA的衰减。这些干预措施对干细胞产生的软骨组织的质量和数量的影响将被评估。总而言之，该项目的成果将是鉴定调节SOX 9基因的因素，定义使其易于受到这种调节的基因的特征，并评估这些发现对提高我们工程软骨组织能力的潜力。在提供这些成果的过程中，该项目旨在更好地了解关键软骨基因的基本调控，这将影响我们如何理解与年龄相关的组织病理学并改善组织再生技术。