High-throughput Sequencing Analysis Of Osteogenesis-related MiRNAs In Osteoclast-derived Exosomes Under Simulated Microgravity Condition

During long-term space flight,space bone loss is the primary risk factor which seriously threatens the health of astronauts.Bone loss is induced by regulation of astronauts’ bone metabolism imbalance in microgravity condition.Osteoclasts play a key role in the regulation of bone metabolism,the regulation mechanism of osteoclasts on osteoblasts is very important in the field of bone metabolism research.In recent years,as a new way of cell-to-cell communication,exosomes have attracted extensive attention from researchers at home and abroad.Meanwhile,micro RNAs(miRNAs)as a signaling molecule are involved in the osteoclasts-osteoblasts communication.In this process,the miRNA was encapsulated into exosomes to directly mediate cellular communication.However,what is the effect of osteoclastderived exosomes on the activity and function of osteoblasts in microgravity condition? Does the exosomes contain miRNAs regulatory molecules involved in osteoblast bone formation?What is the molecular mechanism of these miRNAs regulating bone formation? The clarification of these questions should be one of the important breakthroughs in explaining how osteoclasts regulate osteoblasts under microgravity condition.Therefore,this study was started with osteoclast-derived exosomal miRNAs.Random positioning machine was used as simulated microgravity model for cell culture.And RANKL was used to induce pre-osteoclast RAW264.7 cells to differentiate into mature osteoclasts and extract its exosomes to investigate the effect of osteoclast-derived exosomes on osteoblasts.High-throughput sequencing was performed to reveal miRNAs’ expression profiles of osteoclast-derived exosomes.Osteogenesis-related miRNAs were screened for the further study.In addition,bioinformatics analysis tools were used to predict target genes,and GO function and KEGG pathway enrichment analysis were carried out for candidate target genes.Finally,real-time quantitative PCR(RT-q PCR)was used to verify osteogenesis-related miRNAs sequencing results.The results suggested that osteoclastogenesis of RAW264.7 cells was enhanced by the RPM system.A greater number of giant TRAP-positive multinucleated cells were observed in the RPM group(RPM)than in the normal gravity control group(CON)cell cultures,which had fewer and smaller multinucleated cells.Both flow cytometry and Western blot showed that osteoclast-derived exosomes highly expressed CD63 and CD81 surface markers.Scanning electron microscopy and particle size detection experiments showed that exosomes had a particle size of about 80 nm and uniform distribution.It was confirmed by PKH67 staining experiments that MC3T3-E1 cells can take up osteoclast-derived exosomes,and exosomes were added to the MC3T3-E1 cell culture system.Exosomes significantly inhibited proliferation of MC3T3-E1 cells in a dose-dependent manner by the CCK8 assay.Compared with OC-ExosControl,the alkaline phosphatase(ALP)activity of MC3T3-E1 cells was significantly decreased in RPM-OC-Exos.The cell cycle was arrested in the G0 / G1 phase,and the mineralized nodules formed by the MC3T3-E1 cells were significantly reduced.In addition,RPM-OC-Exos can induce early and late apoptosis of MC3T3-E1 cells and further downregulate m RNA expression levels of osteogenic differentiation-related genes ALP,BSP,OCN,OPN,Col Iα1 and Runx2.In order to study the important role of osteoclast-derived exosomes in the regulation of osteoclasts on osteoblasts,the expression profiles of osteoclast-derived miRNAs in CON and RPM groups were further evaluated.The consensus sequence between the CON and RPM groups was 14.6%.755 and 694 of the known miRNAs were identified in the osteoclast-derived exosomes of the CON and RPM groups,and 508 miRNAs were simultaneously identified between CON and RPM groups.Differential expression analysis of known miRNAs was performed by small RNA classification annotation and sequence alignment,differential heat maps were drawn,and new miRNAs were predicted.According to |log2(Fold-change)|≥1 and P value<0.05,116 miRNAs with significant differential expression were screened,of which 29 were up-regulated and 87 were down-regulated.Finally,based on the expression profiles of osteoclast-derived exosomal miRNAs,10 differentially expressed miRNAs were screened in this study.The results of cluster analysis showed a better intra-group repeatability.Candidate target genes(CTGs)of each miRNA were identified by Target Scan,mi RDB,mi RTar Base and mi RWalk online databases.The GO functional analysis determined the Biological Process,Cellular Components(CC)and Molecular Function(MF)of CTGs.The bone metabolism-related signaling pathways enriched by the KEGG pathway are mainly Wnt,TGF-β,MAPK,Notch and m TOR.miRNAs expression were verified by RT-q PCR,and the verification results were highly consistent with the miRNA sequencing results.In summary,this study aims to investigate the effect of osteoclast-derived exosomes on osteoblasts under simulated microgravity condition.High-throughput sequencing technology was used to reveal the osteoclast-derived miRNAs,and the miRNAs expression profile was constructed.One hundred and sixteen significant differentially expressed miRNAs were found,and 10 osteogenesis-related miRNAs were further screened.And candidate miRNAs were subjected to target gene prediction and bioinformatics analysis.The results of this study help to understand the important role of osteoclast-derived exosomal miRNAs in osteoclast-osteoblast communication at the transcriptional level.In order to further elucidate the molecular mechanism of osteoclasts regulating osteoblasts through the paracrine mechanism and provide new ideas and experimental basis for the prevention and treatment of space bone loss.