Regulation of microRNA homeostasis: Implications in bone fracture healing

摘要 骨折愈合受损或延迟是一个临床问题，在美国每年影响超过150万人; 肥胖和相关的2型糖尿病（T2 D）是在此背景下的重要且独立的风险因素。 近34%的美国人口肥胖，预计这一数字在未来将大幅攀升。 十年因此，肥胖/T2 D相关骨折愈合受损的发生率将不断增加。 关心尽管有这些发人深省的统计数据，肥胖/T2 D延迟愈合的分子基础仍然存在 未知，并要求调查。最近，已经确定饮食中的骨折愈合受损- 诱导肥胖（DIO）小鼠模型，其是肥胖和高血糖症的已建立模型， 伴随着骨折骨痂内脂肪细胞数量的增加。我们对这些研究进行了跟踪， 发现葡萄球菌核酸酶和tudor结构域1（Tudor-SN，缩写为TSN） 促进小鼠原代骨髓间充质干细胞（BMSC）的脂肪生成，以及 在小鼠3 T3-L1和人HprAD前脂肪细胞中，通过降解特定的抗脂肪形成microRNA 包括两种抑制过氧化物酶体增殖物激活受体表达的关键miRNAs γ（PPARg），脂肪生成的主要调节剂。值得注意的是，我们还发现TSN表达是 与瘦小鼠相比，DIO小鼠的骨痂内的PPARg局部升高，与编织- 骨衬细胞，并且发生在紧接脂肪细胞开花之前的时间点。下调 通过局部递送化学修饰的TSN siRNA的愈伤组织TSN抑制脂肪生成，并增强 DIO小鼠中的矿化愈伤组织形成。根据这些初步数据，我们建议中央 假设TSN是肥胖/T2 D中发生的延迟骨愈合的关键分子介质。到 为了验证这个假设，我们提出了两个具体目标。在第一个具体目标中，我们将阐明 TSN作为BMSC分化的调节剂和促进周转的脂肪形成的介质的作用 抗脂肪形成的miRNAs。我们将使用RNA-seq、miR-seq和RT-qPCR来研究TSN敲除的效果。 对从野生型和TSN敲除小鼠分离的原代BMSC中的mRNA和miRNA库的影响。在 第二个具体目标，我们将表征TSN在DIO小鼠延迟骨愈合中的功能。我们将首先 比较骨折愈合相关基因在不同愈合阶段的表达水平， DIO小鼠。将使用RNA-seq、RT-qPCR和多重PCR在转录组水平上进行比较。 荧光原位杂交，并在蛋白质水平上使用免疫荧光染色。我们还将 使用miR-seq和RT-qPCR比较miRNA的表达水平。最后，我们将研究 在瘦小鼠和DIO小鼠的不同愈合阶段向骨折骨痂递送TSN siRNA。完成 所提出的实验将使我们能够确定TSN及其靶向miRNA在肿瘤发生过程中的作用。 骨折修复并与肥胖/T2 D相关的骨折愈合受损有关的TSN。

Abstract Impaired or delayed fracture healing is a clinical problem that affects >1.5 million people in the US annually; obesity and associated type 2 diabetes (T2D) are significant and independent risk factors in this context. Nearly 34% of the US population is obese, and the number is projected to climb significantly in the coming decade. Therefore, the incidence of obesity/T2D-associated impaired fracture healing will be a growing concern. Despite these sobering statistics, the molecular basis for delayed healing in obesity/T2D remains unknown and begs investigation. Recently, it has been established that impaired fracture healing in the diet- induced obesity (DIO) mouse model, which is an established model of obesity and hyperglycemia, is accompanied by an increased number of adipocytes within fracture callus. We followed up on these studies to discover that Staphylococcal nuclease and tudor domain-containing 1 (Tudor-SN, abbreviated as TSN) promotes adipogenesis in murine primary bone marrow-derived mesenchymal stem cells (BMSCs), as well as in mouse 3T3-L1 and human HprAD preadipocytes, via degrading particular anti-adipogenic microRNAs (miRNAs), including two key miRNAs that inhibit the expression of peroxisome proliferator-activated receptor gamma (PPARg), the master regulator of adipogenesis. Remarkably, we also found that TSN expression is focally elevated within the callus of DIO mice compared to lean mice, co-localizing with PPARg in the woven- bone lining cells, and occurring at time points immediately preceding the adipocyte bloom. Downregulating callus TSN via local delivery of a chemically modified TSN siRNA inhibited adipogenesis and enhanced mineralized callus formation in DIO mice. According to these preliminary data, we propose the central hypothesis that TSN is a key molecular mediator of the delayed bone healing that occurs in obesity/T2D. To test this hypothesis, we propose to execute two Specific Aims. In the first Specific Aim, we will elucidate the role of TSN as a regulator of BMSCs differentiation and a mediator of adipogenesis that promotes the turnover of anti-adipogenic miRNAs. We will use RNA-seq, miR-seq, and RT-qPCR to study the effect of TSN knockout on the mRNA and miRNA pools in primary BMSCs isolated from wild-type and TSN knockout mice. In the second Specific Aim, we will characterize TSN function in delayed bone healing in DIO mice. We will first compare the expression levels of fracture healing-associated genes at various stages of healing in lean and DIO mice. Comparisons will be performed on the transcriptomic level using RNA-seq, RT-qPCR, and multiplex fluorescence in situ hybridization, and on the protein level using immunofluorescence staining. We will also compare the expression levels of miRNAs using miR-seq and RT-qPCR. Finally, we will study the impact of delivering a TSN siRNA to the fracture callus on different stages of healing in lean and DIO mice. Completion of the proposed experiments will enable us to define the role of TSN and its target miRNAs in the process of fracture repair and implicate TSN in obesity/T2D-associated impaired fracture healing.