Regulation of microRNA homeostasis: Implications in bone fracture healing

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.