The Effect Of MiR-503on Regulating The Differentiation Of Osteoclast And Thus Modulating Postmenopausal Osteoporosis

Section One:MiR-503is dramatically down-regulated in CD14+PBMCs of postmenopausal osteoporosis subjectsAims:To separate and purify CD14+peripheral blood mononuclear cells (PBMCs) from postmenopausal osteoporosis subjects and postmenopausal women with normal bone mass respectively; To investigate the expression profile of PBMCs microRNA of postmenopausal osteoporosis subjectsMethods:We recruited51Chinese postmenopausal women. Among them,26are diagnosed as oesteporosis and the other25have normal bone mass. From each group,5subjects were randomly selected as subgroups for investigation in both molecular an cellular levels. In each subgroup, we first separated the PBMCs by the method of density gradient centrifugation, and selected those with highly purified CD14+PBMCs using by means of immunomagnetic cell sorting. Then we compared the miRNA expression profile of PBMCs between subgroups by using the miRNA microarray technology. Subjects with significant decrease in miR-503expression were identified for further investigation. Finally, we verified the expression profile of MiR-503in PBMCs from all51subjects using qRT-PCR.Results:(1) Among all the subjects, twenty-six meet the diagnosis standard for osteoporosis and the other25have normal bone mass. Both groups are similar in age, weight and serum estradiol, showing no significant differences in calcium intake, years of postmenopause, physical exercise, PTH levle,25-hydroxyvitaminD and TNF-a. However, bone-specific alkaline phosphatase (BAP), a bone-formation marker, significantly decreases in the osteoporosis group comparing to the normal control (P<0.05).(2) We successfully separated mononuclear cells from the peripheral blood in both groups by the method of density gradient centrifugation and then harvested highly purified CD14+PBMCs by means of immunomagnetic cell sorting (3) With the technology of mircoRNA microarry, we detected miRNA expression profile of CD14+PBMCs from both groups, and successfully identified five miRNAs whose expression is different in each group. Comparing with the normal control, the expressions of Hsa-miR-503, hsa-miR-218, and hsa-miR-618decrease significantly, while hsa-miR-133a and hsa-miR-107were upregulated significantly in the postmenopausal osteoporosis group. The expression of hsa-miR-503was downregulated most.(4) Using PCR technology, we found that MiR-503expresses in all51subjects and they were consistent with those detected by miRNA microarray from the subgroups, i.e., the expression of MiR-503significantly lower in menopause women with osteoporosis compared to the normal control. Conclusions:We identified that5miRNAs were differently expressed in the CD14+PBMCs from both subgroups. Hsa-miR-503, hsa-miR-218, hsa-miR-618were downregulated in postmenopausal osteoporosis subgroup, while hsa-miR-133a, hsa-miR-107were upregulated. As the most dramatically downregulated miRNA, we chose miR-503as object and investigated its function during osteoclast differentiation. The expression of miR-503was detected in all51subjects using PCR methods and the results were consistent with that of miRNA microarray. Section two:MiR-503regulates osteoclastogenesis and involved in OVX mice osteoporosisAims:To investigate the role of has-miR-503in the osteoclast differentiation of CD14+PBMCs at the cellular level; To investigate the effect of has-miR-503on bone at the animal level.Methods:At the cellular level, CD14+PBMCs from the normal control group were first transfected with pre-miR-503and antagomiR-503respectively. After we detected the expression level of matured miR-503using Northern Blot, CD+14PBMCs were induced to differentiate into osteoclast by M-CSF and RANKL. During this process, both Nuclear factor of activated T-cells cytoplasmic (NFATc1)--the key transcription factor for osteoclast differentiation--and the mRNA level of TRAP--the specified gene of osteoclast--were quantitatively measured by PCR in realtime; Meanwhile, we examined the status of osteoclast differentiation by TRAP staining。The same protocol was applied to the CD14+PBMCs from the osteoporosis subgroup. Namely, after being transfected with pre-miR-503to restore the expression of miR-503, CD14+PBMCs were exposed to M-CSF and RANKL to induce the differentiation into osteoclast. Also the mRNA level of both osteoclast differentiation indexes, TRAP and NFATc1, were measured by realtime quantitative PCR and TRAP staining was used to count the multi-nuclear giant cells. In vivo, we used the OVX to mimic menopause, and SHAM groups as the control. In boh OVX and SHAM groups, ago-mir503or antagomiR-503were used to stimulate or inhibit the expression of mir-503in vivo. Dual-energy X-ray absorptiometry (DXA) were used to detect the BMD in neck of femur, and MicroCT were used to detect the microstructure of bones. Meanwhile, the mRNA level of TRAP and NFATc1were measured by realtime quantitative PCR.Results:(1) We successfully constructed the has-miR-503expression vector-pre-miR-503using pSilncer4.1-CMV puro so that has-miR-503overexpessed in PBMCs transfected with pre-miR-503. On the contrary, the expression of has-miR-503was inhibited in PBMCs transfected with antagomiR-503.(2) In PBMCs from the normal control, the overexpression of has-miR-503inhibited the formation of TRAP+multinuclear giant cells and lower the mRNA levels of TRAP and NFATC1. On the contrary, the inhibition of has-miR-503by antagomiR-503enhanced all the osteoclast differentiation indicators significantly. In PBMCs from the osteoporosis subgroup, the restoration of has-miR-503expression can inhibit the formation of TRAP+multinuclear giant cells, and lower the mRNA levels of TRAP and NFATC1.(3) In the animal models, ago-mir503stimulated the overexpression of miR-503and antagomiR-503inhibited its expression.(4) In vivo, the overexpression of mir-503in SHAM mice increases the bone density and improves the microstructure of bones. Vice versa, the inhibition of has-miR-503decreases the bone density and destroys the microstructure of bones. While, restoration of miR-503inhibited bone resorption and prevented bone loss in OVX miceConclusion:(1) In menopause women with normal bone mass, the overexpression of has-miR-503inhibits the differentiation of CD14+PMBCs into osteoclasts; the inhibition of has-miR-503promotes the differentiation of CD14+PMBCs into osteoclasts. In menopause women with osteoporosis, the restoration of miR-503expression is able to stop the differentiation of CD14+PMBCs into osteoclasts.(2) In vivo, silencing of miR-503in SHAM mice promoted bone resorption and decreased bone mass. While, restoration of miR-503inhibited bone resorption and prevented bone loss in OVX mice. Section three:The mechanism of how has-miR-503regulates the differentiation of osteoclastAims:To predict and verify the target gene of has-miR-503, so that we could understand the mechanism of how has-miR-503regulates the differentiation of CD14+PBMCs into osteoclasts.Methods:First, The RNA22target gene prediction software was used to predict the target gene of has-miR-503. Second, both M-CSF and RANKL were used to induce the differentiation of CD14+PBMCs in both normal control and osteoporosis subgroups. The mRNA levels of target RNAK and mir-503were measured by Northern blot and the RNAK protein levels were measured by Western blot. The expression profiles of both Mir-503and RANK in the differentiation of osteoclasts were compared between two subgroups. Third, we cloned the Coding sequence (CDS) section of the target gene RANK which contains the has-miR-503binding site to the CDS carried by pGL3to construct the wild type carrier WT-pGL3-RANK. Meanwhile, we constructed the mutation carrier MUT-pGL3-RANK using the QuickChange site-directed mutagenesis kit. These two reporter vectors were cotransfected with pre-miR-503or miR-C into CD14+PBMCs, and Dual Luciferase Reporter Assay System was used to detect the luminescent signal. Finally, we trasfected CD14+PBMCs with pre-miR-503antago-miR-503 respectively. The mRNA level and protein level of the target RANK gene was detected by realtime quantitative PCR and Western blot respectively to verify the regulation of has-miR-503on the target gene.Ressults:(1) The RNA22-target-gene-prediction software predicted that the target gene of has-miR-503is RANK.(2) In menopause women with normal bone mass, when CD14+PBMCs differentiated into osteoclast, miR-503was slightly upregulated; RANK mRNA significantly increased but RANK protein did not change. In the osteoporosis subgroup on the other hand, the miR-503expression in CD14+PBMCs is significantly lower than the normal control group at the basal level. When CD14+PBMCs differentiated into osteoclasts, both the mRNA and protein expression of RANK increases.(3)Compared with the control group transfected with miR-C, the activity of WT-pGL3-RANK was significantly inhibited after being co-transfected with pre-miR-503. However, cotransfection with pre-miR-503, MUT-pGL3-RANK abolished this repression.(4) Transfection by pre-miR-503lowers the protein expression of RANK but has little effect on its mRNA; transfection by antagomiR-503alone increases the RANK protein expression without changing its mRNA level. Therefore, we concluded that has-miR-503inhibits the differentiation of osteoclast mainly by inhibiting the protein expression of RANK. Conclusions:(1) RANK is the target gene of has-miR-503, which was predicted by the bioinformatics software and further verified by Luciferase reporter vector.(2) In both groups, during the differentiation process of CD14+PBMCs into osetoclasts, the expression profiles of miR-503and RANK are consistent with that of the microRNA on its target genes.(3) At the cellular level, has-miR-503inhibits the RNAK expression at the transcription level, preventing the osteoclast differentiation.