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，每年影响美国150万人； 在这种情况下，肥胖和相关的2型糖尿病(T2D)是重要的和独立的危险因素。 近34%的美国人患有肥胖症，预计这一数字在未来还会大幅攀升 十年。因此，肥胖/T2D相关的骨折愈合受损的发生率将会不断增长 担忧。尽管有这些发人深省的统计数据，但肥胖症/T2D延迟愈合的分子基础仍然存在 不明身份并请求调查。最近，人们发现饮食中的骨折愈合障碍-- 诱导肥胖(DIO)小鼠模型是一种已建立的肥胖和高血糖模型。 伴有骨折骨痂内脂肪细胞的增多。我们跟进了这些研究，以 发现葡萄球菌核酸酶和含有Tudor结构域的1(Tudor-SN，缩写为TSN) 促进小鼠原代骨髓间充质干细胞(BMSCs)的脂肪生成 在小鼠3T3-L1和人HprAD前体脂肪细胞中，通过降解特定的抗造脂microRNAs (MiRNAs)，包括抑制过氧化物酶体增殖物激活受体表达的两个关键miRNAs 伽玛(PPARg)，脂肪形成的主要调节因子。值得注意的是，我们还发现TSN的表达是 与瘦小鼠相比，DIO小鼠的骨痂内局部升高，与PPARg共同定位于编织的 骨衬里细胞，发生在紧接脂肪细胞盛开之前的时间点。下调监管 经化学修饰的TSN siRNA局部导入的愈伤组织TSN抑制脂肪生成并增强 DIO小鼠的矿化骨痂形成。根据这些初步数据，我们建议中央 假设TSN是肥胖/T2D延迟骨愈合的关键分子介质。至 检验这一假设，我们提出了执行两个具体目标。在第一个具体目标中，我们将阐明 TSN作为骨髓间充质干细胞分化调节因子和促进转化的脂肪生成介质的作用 抗脂肪生成的miRNAs。我们将使用rna-seq、miR-seq和RT-qPCR来研究TSN基因敲除的效果。 从野生型和TSN基因敲除小鼠分离的原代BMSCs的信使核糖核酸和miRNA池。在 第二个特定目的，我们将研究TSN在DIO小鼠延迟骨愈合中的作用。我们将首先 比较不同愈合阶段骨折愈合相关基因的表达水平 迪奥老鼠。我们将使用rna-seq、rt-qpr和多重技术在转录水平上进行比较。 荧光原位杂交，并在蛋白质水平上用免疫荧光染色。我们还将 用miR-seq和RT-qPCR比较miRNAs的表达水平。最后，我们将研究 在LINE和DIO小鼠骨折愈合的不同阶段，将TSN siRNA传递到骨折骨痂。完成 将使我们能够确定TSN及其靶向miRNAs在 骨折修复和TSN在肥胖/T2D相关的骨折愈合受损中的牵连。

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.