Evaluation of osteoclast-derived exosomal miRNA under simulated microgravity conditions using next-generation sequencing
Introduction
During space flight, it has been documented that astronauts suffer from progressive bone loss, which has become a major concern to their health [[1], [2], [3]]. The bone mass of astronauts has revealed a significant reduction in their total bone mineral density with a loss of 1–2% per month under microgravity conditions [4]. The potential mechanism of space bone loss is thought to be the result of an uncoupling of bone remodeling in microgravity [5,6]. Bone remodeling relies on the tightly regulated communication between bone-forming osteoblasts and bone-resorptive osteoclasts. Several studies have focused on the functional alteration of osteoclasts and osteoblasts in microgravity [[7], [8], [9], [10], [11]].
Numerous studies have demonstrated that microgravity can directly stimulate osteoclastogenesis and increase bone resorption in space or in a simulated microgravity environment [[12], [13], [14]]. These findings indicate that osteoclasts and their precursors are the direct targets of mechanical forces. However, the cellular and molecular mechanisms of osteoclast behavior in microgravity need to be further elucidated. Bone cell communication is dependent on the transportation of key bone cell-derived factors, including membrane-bound ligands, receptors, diffusible paracrine factors and extracellular vesicles (EVs) [[15], [16], [17], [18], [19]]. EVs are common membrane-surrounded structures released by various mammalian cells in an evolutionarily conserved manner [20]. The EV class includes exosomes, microvesicles, and apoptotic bodies [21,22]. Exosomes, nano-sized extracellular vesicles (30–150 nm), are released from various cells in response to the surrounding environment, external stimuli, and physiological status of the cell, and play a potential role in intercellular communication [23,24]. Moreover, exosomes have been considered a critical factor in bone remodeling [25,26]. Bone cell-derived exosomes carry specific biochemical cargo, encompassing bioactive proteins, lipids, metabolites, mRNA, microRNAs (miRNAs) and many other non-coding RNAs, to regulate the functions of target cells [27]. Recent studies have demonstrated that osteoclast-derived exosomes represent a novel approach for osteoclast-osteoblast communication by delivering genetic information for bone remodeling [25,28]. Previous work by our group has shown that osteoclast-derived exosomes have a negative-feedback mechanism between osteoclasts and osteoblasts under simulated microgravity conditions [29].
miRNAs are a superfamily of small single-stranded non-coding RNAs (approximately 20–25 nucleotides in length) that can negatively regulate gene expression at the post-transcriptional level by binding to the 3′-untranslated region (3′-UTR) of target mRNAs, causing translational repression of mRNA [30]. miRNAs can be released into the microenvironment via exosomes and exert important functions in cellular communication [31]. To date, several studies have suggested that miRNAs, such as miR-214, miR-148a, miR-680 and miR-1192, are associated with bone remodeling by regulating the proliferation and differentiation of osteoclasts and osteoblasts [26,28,32,33]. Therefore, the transfer of miRNAs by osteoclast-derived exosomes is specifically important for osteoclast-osteoblast communication. However, the role and miRNA expression profiles of osteoclast-derived exosomes in microgravity remain largely unknown.
Based on these results, we hypothesized that miRNAs derived from osteoclasts might represent an important role in osteoblast-osteoclast communication under microgravity conditions. In this study, the random positioning machine (RPM) was used as a ground-based model to simulate microgravity. Exosomes from RAW264.7 cell-derived osteoclasts were obtained from this RPM system. By using miRNA next-generation sequencing (NGS), we further evaluated exosomal miRNA profiles under microgravity conditions compared with normal gravity conditions. The purpose of this study was to explore the expression patterns of osteoclast-derived exosomal miRNAs under microgravity conditions. This work may provide new insights into the molecular mechanisms of bone loss in microgravity and therapeutic targets to prevent osteoporosis in astronauts during space flight missions.
Section snippets
The facility of a random positioning machine (RPM) system for cell culture
The RAW264.7 murine osteoclastic precursor cell line [34] was used in this study and purchased from the Cell Collection Center of Shanghai, which originally obtained the cell line from the American Type Culture Collection (Manassas, VA, USA). For cell culture, osteoclastic RAW264.7 cells were maintained in α-minimum essential medium (α-MEM; Gibco, Carlsbad, USA) supplemented with 2 mmol/L l-glutamine, 2.2 g/L sodium bicarbonate, 100 units/ml penicillin, 0.1 mg/mL streptomycin and 10% (v/v)
Characterization of osteoclast-derived exosomes
Murine osteoclastic precursor RAW264.7 cells were seeded onto carrier slides for their differentiation until reaching maturity in the normal conditions, then put the slides into the dedicated cell culture flasks and fixed the flasks in the RPM system (Fig. 1A). In this study, we applied an ultracentrifugation method to obtain CON and RPM exosomes from cell culture medium. Exosomes secreted by osteoclasts under normal conditions and in the RPM were successfully isolated and fully characterized
Discussion
Osteoclast-derived exosomes from the RPM group can obstruct the differentiation of MC3T3-E1 cells by interfering with the Wnt/β-catenin signaling pathway, which plays an essential role in the regulation of bone remodeling in a microgravity environment [29]. Most importantly, the reduced bone formation is the primary characteristic of bone loss during space flight, and miRNAs have been demonstrated to regulate gene expression at the post-transcriptional level. These miRNAs which can regulate
Declaration of interest statement
The authors report no conflicts of interest.
Acknowledgments
We gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC) (31500688, 81502465, 81700823 and 51477141), the Fundamental Research Funds for the Central Universities (3102016OQD042), the Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University (Grant No.ZZ2019272).
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These authors contributed equally to this study.