Study Of Serum MiRNAs Under Simulated Microgravity

During long-term space flight, the organism is forced to adapt to the new microgravity environment for the absence of gravity. These adaptive changes of physiology that result from microgravity, including bone loss, cognitive impairment, muscle atrophy and so on, was named space adaptation syndrome and has been a major obstacle of human long-term space exploration.Microgravity seriously impacts the health and mission performance of astronauts. To monitor and counteract these negative effects and reveal the mechanism are the priority of space medical research. Developing simple and effective biomarkers is of great significance for the health monitoring of astronauts. Meanwhile, using a systems biology approach to investigate the dynamic and systematic effect of microgravity will be an effective method in space medical research. In this study, we used rat hindlimb unloading as main animal model to explore the effects of simulated microgravity by serum miRNAome. The main results are summarized as following:Firstly, we performed a systematic study on serum miRNAs to identify suitable reference gene for normalization. miR-25-3p was identified as the suitable reference gene through a large-scale screening using miRNA microarray, qPCR validation and statistical algorithms (geNorm and Normfinder). Further analysis showed that miR-25-3p was highly conserved among species, and its expression showed fairly stable in simulated microgravity model (hindlimb unloading rat and bedrest rhesus monkey) and osteoporosis model (OVX rat and postmenopausal woman). These results confirmed that miR-25-3p was a stable reference gene and provided foundation for the following serum miRNAs research.Then, we screened potential serum miRNAs biomarkers for the postmenopausal osteoporosis and the simulated microgravity-induced osteoporosis. miRNA microarray and qPCR were performed in OVX rats and 15 serum miRNAs were identified differentially expressed. We further detected that miR-30b-5p was significantly downregulated in serum of postmenopausal women with bone loss (osteopenia or osteoporosis), and miR-103-3p, miR-142-3p, miR-328-3p were significantly downregulated in osteoporosis. Multiple correlation analysis showed that these miRNAs were significantly positive correlate with H-BMD, meanwhile, miR-30b-5p and miR-142-3p were significantly associated with FN-BMD. These miRNAs were potential biomarker for postmenopausal osteoporosis. To investigate the potential biomarkers for simulated microgravity induced osteoporosis, we detected these miRNAs in the serum of rhesus monkeys with or without long-duration bedrest. miR-30b-5p, miR-103-3p and miR-142-3p were significantly downregulated after bedrest, while miR-328-3p had no significant change. These three miRNAs were potential biomarkers for simulated microgravity induced osteoporosis and showed considerable diagnostic value by ROC analysis.To gain better insight into the impacts of microgravity on organism, an association analysis was performed on serum miRNAome and metabonomics data to provide clue and direction for the following research. miRNAs microarray and qPCR identified that 13 miRNAs were upregulated in the serum of rat with hindlimb unloading for 4w or 5w and highly correlated with nervous system especially hippocampus by the analysis of Panther and DAVID. Moreover, the serum metabolites showed the significant changes after hindlimb unloading for 5w. The association analysis of serum miRNAome and metabonomics showed that the simulated microgravity had important effect on nervous system especially hippocampus.According to the bioinformatics analysis, we detected the expression of miRNAs, mRNAs and proteins in hippocampus. miR-383-5p was significant upregulated after hindlimb unloading, while its predicted target gene AQP4 showed significant downregulated in both mRNA and protein level. We then confirmed that AQP4 is a target gene of miR-383-5p by dual luciferase reporter experiment in vitro. AQP4 plays important role in water balance of brain and is closely related to the cognitive function of hippocampus. We hypothesized that the downregulated of AQP4 is an underlying mechanism of microgravity impacting cognitive function of hippocampus and may associate with the head hyperemia under microgravity.Taken together, we identified that miR-25-3p is a suitable reference gene for simulated microgravity and osteoporosis model; A group of miRNAs screened in the serum could serve as non-invasive biomarkers in detection of postmenopausal osteoporosis and simulated microgravity-induced osteoporosis; Meanwhile, we explored that the underlying mechanism of microgravity impacts cognitive function of hippocampus.