Regulation of microRNA homeostasis: Implications in bone fracture healing

microRNA 稳态的调节：对骨折愈合的影响

• 批准号: 10194485
• 负责人: Reyad A Elbarbary
• 金额: $ 39.58万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-11 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10194485
• 关键词: Address; Adipocytes; Affect; Biology; Bone Marrow; Bone callus; Cell Differentiation process; Cells; Chemicals; Clinical; Complex; Data; Defect; Development; Fatty acid glycerol esters; Fluorescent in Situ Hybridization; Fracture; Fracture Healing; Gene Expression; Genes; Grant; Histological Techniques; Homeostasis; Human; Hydrogels; Hyperglycemia; Immature Bone; Immunofluorescence Immunologic; Impaired healing; Impairment; In Vitro; Incidence; Investigation; Knock-out; Knockout Mice; Mammalian Cell; Marrow; Measures; Mediating; Mediator of activation protein; Mesenchymal Differentiation; Mesenchymal Stem Cells; Messenger RNA; MicroRNAs; Micrococcal Nuclease; Minerals; Modeling; Molecular; Mus; Musculoskeletal; Non-Insulin-Dependent Diabetes Mellitus; Obese Mice; Obesity; Osteogenesis; PPAR gamma; Pathologic; Pathology; Pathway interactions; Phase; Phenotype; Play; Population; Process; Proteins; Regulation; Reporting; Resources; Risk Factors; Role; Science; Site; Small Interfering RNA; Stains; Testing; Thinness; Time; Tissue-Specific Gene Expression; Type 2 diabetic; United States National Institutes of Health; Work; bone healing; diabetic patient; diet-induced obesity; experimental study; follow-up; genome-wide; genome-wide analysis; healing; high reward; lipid biosynthesis; miRNA expression profiling; mouse model; multipotent cell; nanoparticle; novel; novel therapeutic intervention; nuclease; osteogenic; personalized approach; ranpirnase; repaired; role model; statistics; stem cell differentiation; transcription factor; transcriptome sequencing; transcriptomics

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.

抽象的 受损或延迟的骨折愈合是一个临床问题，每年在美国影响> 150万人； 在这种情况下，肥胖和相关的2型糖尿病（T2D）是重要且独立的风险因素。 美国近34％的人口肥胖，预计该人数将在即将到来的 十年。因此，肥胖/T2D相关的骨折愈合的发生率将在增长 忧虑。尽管这些清醒的统计数据，肥胖/T2D延迟愈合的分子基础仍然存在 未知并乞求调查。最近，已经确定了饮食中骨折愈合受损 - 诱导肥胖（DIO）小鼠模型是肥胖和高血糖的建立模型，是 伴随着骨折愈伤组织中脂肪细胞数量的增加。我们跟进了这些研究 发现葡萄球菌核酸酶和都铎式结构域的1（都铎 - SN，缩写为TSN） 促进鼠原代骨骨髓中的间充质干细胞（BMSC）中的脂肪形成 在小鼠3T3-L1和人Hprad前脂肪细胞中，通过降解特定的抗辅助microRNA （miRNA），包括两个抑制过氧化物酶体增殖受体表达的关键miRNA Gamma（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 与瘦小小鼠相比 骨衬里细胞，并在脂肪细胞开花之前在时间点发生。下调 愈伤组织TSN通过局部递送化学修饰的TSN siRNA抑制脂肪形成并增强 DIO小鼠的矿化愈伤组织形成。根据这些初步数据，我们提出了中央 假设TSN是肥胖/T2D中发生的延迟骨愈合的关键分子介体。到 检验此假设，我们建议执行两个具体目标。在第一个特定目标中，我们将阐明 TSN作为BMSC分化的调节剂和促促进营业额的脂肪生成介质的作用 抗辅助miRNA。我们将使用RNA-Seq，miR-Seq和RT-QPCR研究TSN敲除的效果 从野生型和TSN敲除小鼠中分离出的原代BMSC的mRNA和miRNA池上。在 第二个特定目的，我们将表征DIO小鼠延迟骨愈合中的TSN功能。我们将首先 比较在瘦肉和 Dio小鼠。比较将使用RNA-SEQ，RT-QPCR和多重分数在转录组级别上进行 荧光原位杂交，并使用免疫荧光染色在蛋白质水平上。我们也会 使用miR-Seq和RT-QPCR比较miRNA的表达水平。最后，我们将研究 在瘦小和DIO小鼠的不同阶段，将TSN siRNA传递到骨折愈伤组织中。完成 在拟议的实验中，我们能够定义TSN及其目标miRNA的作用 断裂修复并牵涉到肥胖/T2D相关的骨折愈合受损中的TSN。