MicroRNA Profiling Of Circulating CD14~+Peripheral Blood Mononuclear Cells From Postmenopausal Osteoporosis Patients And The Study Of Methylation Of The MiR-503Promoter

Chapterl microRNA profiling of circulating CD14+peripheral blood mononuclear cells from postmenopausal osteoporosis patientsObjective:To compare the miRNAs expression profiles in the CD14+PBMCs from postmenopausal osteoporosis patients and postmenopausal healthy women.Methods:Firstly, PBMCs were separated from peripheral blood of osteoporosis patients (n=26) and age-matched healthy postmenopausal controls (n=25)by the method of density gradient centrifugation, CD14+PBMCs were purified by means of immunomagnetic cell sorting. Secondly,5subjects of each subgroup were selected randomly to perform microarray assays. Lastly, the result was verified by real time RT-PCR.Results:(1) We identified that5miRNAs were differently expressed in the CD14+PBMCs from both subgroups. Hsa-miR-503, hsa-miR-218, hsa-miR-618were downregulated in osteoporosis subgroup. Reversely, hsa-miR-133a, hsa-miR-107were upregulated. The most dramatically downregulated miRNA was has-miR-503.(2) The expression of miR-503was detected in all51subjects using PCR methods and the results were consistent with that of miRNA microarray.Conclusions:miRNA microarray shows that5miRNAs were differently expressed in the CD14+PBMCs from both subgroups. Hsa-miR-503, hsa-miR-218, hsa-miR-618were downregulated in osteoporosis subgroup. Reversely, hsa-miR-133a, hsa-miR-107were upregulated. The most dramatically downregulated miRNA was has-miR-503. Chapter2Methylation of the miR-503promoter of circulating CD14+peripheral blood mononuclear cells from postmenopausal osteoporosis patientsObjective:To exam miR-503promoter methylation status in CD14+PBMCs of postmenopausal osteoporosis patients. To further explore why miR-503expression was the lowest from postmenopausal osteoporosis women.Methods:A429-bp fragment in the miR-503promoter (position-1897to-1469bp) was analyzed in the CD14+PBMCs of osteoporosis patients (n=26) and age-matched healthy postmenopausal controls (n25) by bisulfite sequencing (BSP).Results:(1) The mean±SD methylation levels of the twelve CpG pairs within the amplified promoter region (positions-1830,-1796,-1759,-1756,-1741,-1719,-1689,-1661,-1590,-1581,-1530,-1509) were significantly higher in the osteoporosis samples than in the healthy control samples (0.8±0.2vs.0.51±0.16, P<0.01).(2) The overall methylation levels of the twelve CG pairs within the promoter fragment were inversely correlated with miR-503expression in CD14+PBMCs cells of osteoporosis patients and healthy controls (r-0.942, P<0.001).Conclusions:(1) The methylation status of the region studied was higher in CD14+PBMCs of postmenopausal osteoporosis patients than the postmenopausal healthy controls.(2) miR-503promoter methylation level was negatively correlated with the miR-503expression level in CD14+PBMCs of postmenopausal osteoporotic women and postmenopausal healthy women.