Screening And Identification Of Osteoporosis-associated MiRNAs And Their Response To Simulated Weightlessness

Weightlessness osteoporosis is one kind of disuse osteoporosis induced by the loss of gravity.There is no effective measures to restrain weightless bone loss for the shortage of clear guidance of certainty mechanism.Therefore,it is desirable to study the mechanism of weightless bone loss and find a new therapeutic target for weightless osteoporosis.Mi RNAs are a class of small noncoding RNAs that modulate gene expression via posttranscriptional modification.They play an important role in bone formation and resorption by regulating the formation of osteoclast and the differentiation of osteoblast.The alteration in mechanical load causes the variation of miRNA expression both in bone tissues and osteoblasts,thus affecting osteoblast differentiation and bone formation in microgravity environment.The current research shows that mechanosensitive miRNA was not only involved in development of age-related osteoporosis,but also related to weightless osteoporosis.Based on the above information,the screening of miRNAs related to osteoporosis was firstly explored in this study by multiple models,such as bone tissue and serum from ground population with agerelated osteoporosis,aging and OVX mice models.According to the screening,the miRNAs related to osteoporosis were selected to investigate its response to simulated weightlessness and its role in bone formation in vitro for finding miRNAs involved in development of weightless osteoporosis.This will provide more significant information on theoretical support for the diagnosis and therapy of weightless osteoporosis,and the main results are as follows:(1)Screening,validation and bioinformatics analysis of miRNA related to osteoporosisThe bone tissue of elderly women with osteoporosis was collected from hospitals.Mi RNAs Gene Chip and real-time PCR were used to screen differently expressed miRNAs in bone tissues of osteoporotic women ages 60–69 years and 80–89 years.There were 862 miRNAs captured in bone tissue samples by miRNAs Gene Chip.Twenty-four miRNAs showed the highest differential expression in bone tissues of osteoporotic women in the initial screening.Among the twenty-four miRNAs,four miRNAs were identified in bone tissue of validation cohort by real-time PCR.In comparison with the 60-69 years group,miR-181c-5p and miR-497-5p were downregulated and miR-1290 and miR-204-3p were upregulated in 80–89 years group.Targets of the four miRNAs involve in many signaling pathway associated with osteoblast differentiation and bone metabolism,such as Fox O signaling pathway,estrogen signaling pathway,m TOR signaling pathway,T cell receptor signaling pathway,osteoclast differentiation,insulin signaling pathway,Wnt signaling pathway,MAPK signaling pathway,Hippo signaling pathway,PI3K-Akt and TGF-beta signaling pathway.Some of common target genes for miRNA-181c-5p,and miR-497-5p are involved in osteoblast differentiation and osteoclast formation,such as LRP6,NOTCH2,SMAD7,SOX6,WIF1 and TGIF2.These results suggested that miRNAs expression in bone tissue of elderly osteoporosis was different with different ages.Mi R-181c-5p and miR-497-5p may be closely related to osteoblast differentiation and bone formation,and may also be involved in osteoclast differentiation and bone resorption.(2)Relationship between osteoporosis-related miRNAs and the development of osteoporosis.Based on the above results,the expression of 4 regulated miRNAs(miR-181c-5p,miR-497-5p,miR-204-3p,and miR-1290)were assessed in serum of validation cohort,which consisted of healthy premenopausal women and postmenopausal women with osteopenia and osteoporosis.The effect of anti-osteoporosis drug therapy on their expression level in serum of postmenopausal women with osteopenia or osteoporosis was investigated.Correlation analysis of these miRNAs with BMD,bone turnover,and demography was analyzed.And the regulated miRNAs were further validated in aging and OVX mice models.It was found that miR-181c-5p and miR-497-5p showed downtrend among groups of healthy premenopausal women and postmenopausal women with osteopenia and osteoporosis,and responded to the antiosteoporosis therapy with bisphosphonate plus calcitriol.Mi R-204-3p both in osteopenia and osteoporosis groups were significantly higher than healthy premenopausal women,while there was no difference between osteopenia and osteoporosis groups.Mi R-1290 showed no significant difference among groups including health,osteopenia and osteoporosis groups.And miR-204-3p and miR-1290 had no difference between non-treatment and treatment groups.Besides,miR-181c-5p and miR-497-5p might discriminate patients with osteopenia or osteoporosis from normal bone mass.Moreover,miR-181c-5p and miR-497-5p showed downtrend with aging,and decrease in OVX compared with sham-operated mice.It suggests that miR-181c-5p and miR-497-5p might involve in osteoporosis,and relate to bone loss in progress of osteoporosis.(3)The response and effects of miR-181c-5p and miR-497-5p to simulated weightlessness and on regulation of bone formation in vitro The response of miR-181c-5p and miR-497-5p to changes of mechanical unloading in vivo and in vitro was validated.And the effect of miR-181c-5p and miR-497-5p on osteoblasts and osteoclasts was investigated.The results showed that miR-181c-5p were significantly downregulated in bone tissues of hind limp unloading mouse,while increased after reloading.Mi R-497-5p showed decreased trend,while there was no difference after reloading.In RPM condition,which was used to simulate weightlessness,the expression of miR-181c-5p in MC3T3-E1 cells was upregulated,while the expression of miR-497-5p decreased.All of osteoblasts and osteoclasts expressed miR-181c-5p and miR-497-5p.Expression of miR-181c-5p and miR-497-5p were increased during osteoblastic differentiation,but decreased during osteoclastic differentiation.Exosomes derived from osteogenic differentiated osteoblast were rich in miR-181c-5p and miR-497-5p.Furthermore,overexpression of miR-181c-5p or miR-497-5p promoted osteogenic differentiation of osteoblasts and formation of calcium deposition nodules.In conclusion,this study found that miR-181c-5p and miR-497-5p are involved in bone metabolism and associated with progressive bone loss of osteoporosis.Circulating miR-181c-5p and miR-497-5p might act as potential biomarkers for monitoring treatment effects on osteoporosis and diagnostic approach for osteoporosis.In addition,miR-181c-5p and miR-497-5p are sensitive to changes of mechanical environment,associated with weightlessness-related bone loss,and promote osteogenic differetntaion of osteoblasts and formation of calcium deposition nodules.The results provided experimental basis and theoretical support for the diagnosis and treatment of weightless bone loss.