MicroRNA Profiling During Osteoclast Differentiation And The Functional Study Of MiR-148a

Part one Study on the microRNA profile during the differentiation of human peripheral blood mononuclear cells into osteoclastsObjectiveTo establish the differentiation cell model of osteoclasts derived from human peripheral blood mononuclear cells (PBMCs). To study the microRNA (miRNA) expression profile during the differentiation of PBMCs into osteoclasts.MethodsFirstly, PBMCs were separated from peripheral blood of healthy donors by the method of density gradient centrifugation, CD14+PBMCs were purified by means of immunomagnetic cell sorting. Secondly, the isolated PBMCs were incubated on coverslips or bone slices and differentiated into osteoclasts in the presence of recombinant human macrophage-colony stimulating factor (rh-MCSF) and human receptor activator of nuclear factor-?B ligand (hRANKL). Thirdly, the growth and function of osteoclasts were assessed by tartrate-resistant acid phosphatase (TRAP) staining. The bone slices were stained for toluidine blue to observe bone resorption by osteoclasts. The differentiation cell model of osteoclasts derived from PBMCs was established. Lastly, the differentiated miRNA expression profile during the differentiation of PBMCs into osteoclasts was screened by miRNA microarray and verified by real time RT-PCR.Results(1) CD14+ PBMCs were successfully separated and purified from peripheral blood by means of density gradient centrifugation and immunomagnetic cell sorting.(2) After 14 days of culture of the CD 14+ PBMCs, TRAP+ multinucleated giant cells appeared, and bone slices resorption lacunae could be observed by the staining of toluidine blue.(3) In the present study, miRNA microarray shows that 27 miRNAs were differently expressed during the differentiation of PBMCs into osteoclasts. hsa-miR-148a, hsa-miR-483, hsa-miR-223, hsa-miR-21, hsa-miR-214 were up-regulated, reversely, hsa-miR-155, hsa-miR-125a, hsa-miR-27b, hsa-miR-145 were down-regulated.(4) The result of real time RT-PCR is consistent with that of miRNA microarray, which shows accuracy of real time RT-PCR for verification of differented miRNA expression.ConclusionThe differentiation cell model of osteoclasts derived from PBMCs was established successfully. miRNA microarray shows that 27 miRNAs were differently expressed during the differentiation of PBMCs into osteoclasts. hsa-miR-148a, hsa-miR-483, hsa-miR-223, hsa-miR-21, hsa-miR-214 were up-regulated, reversely, hsa-miR-155, hsa-miR-125a, hsa-miR-27b, hsa-miR-145 were down-regulated. hsa-miR-148a was the most dramatically upregulated miRNA. Part Two Study on the effect of hsa-miR-148a in osteoclast differentiationObjectiveTo investigate the function of hsa-miR-148a during the differentiation of CD14+ PBMCs into osteoclasts.MethodsThe hsa-miR-148a expression profile was detected by Northern blotting during the rh-MCSF and hRANKL induced osteoclastogenesis of CD 14+PBMCs. We designed primers according to the precursor sequence of hsa-miR-148a, and constructed the expression vector of hsa-miR-148a by using the pSilencer 4.1-CMV puro vector. The expression vector was named " pre-miR-148a". After the transfection of CD 14+ PBMCs with pre-miR-148a, osteoclast differentiation was induced by the addition of rh-MCSF and hRANKL. Northern blotting analysis detected the expression profile of hsa-miR-148a. The growth and function of osteoclasts were assessed by TRAP staining and the forming of bone resorption lacunae. The TRAP?NFATc1?OSCAR?CathK mRNA levels were detected by quantitative real-time PCR (qRT-PCR) and the TRAP protein level was detected by Western blotting analysis. Then, we transfected rh-MCSF and hRANKL induced CD 14+ PBMCs with 2'-O-methyl antisense inhibitory oligoribonucleotides (anti-miR-148a). The growth and function of osteoclasts were assessed by TRAP staining and the forming of bone resorption lacunae. The TRAP. NFATc1. OSCAR. CathK mRNA levels and the TRAP protein level were measured as described above.Results(1) The expression of hsa-miR-148a in CD 14+PBMCs was detected after treatment with rh-MCSF and hRANKL and increased progressively with time. (2) High expression of hsa-miR-148a was obtained in CD14+ PBMCs after transfection with the expression vector of hsa-miR-148a constructed by using pSilencer 4.1-CMV puro vector. (3) Compared with control cells, hsa-miR-148a overexpression promotes the formation of TRAP+ multinucleated giant cells and bone slices resorption lacunae. Transfection of pre-miR-148a promoted the mRNA levels of TRAP?NFATc1?OSCAR?CathK. The TRAP protein level was also enhanced by transfection of pre-miR-148a. (4) Transfection of anti-miR-148a inhibited the formation of TRAP+ multinucleated giant cells and bone slices resorption lacunae, also reduced the mRNA levels of TRAP. NFATc1?OSCAR?CathK. The TRAP protein level was also reduced by transfection of anti-miR-148a.ConclusionThe expression vector of hsa-miR-148a was constructed. hsa-miR-148a overexpression promoted osteoclast differentiation of CD 14+ PBMCs. Inhibition of hsa-miR-148a attenuated osteoclast differentiation of CD 14+ PBMCs. Part Three Study on the mechanism of hsa-miR-148a regulating osteoclast differentiationObjectiveTo predict and verify the target gene of hsa-miR-148a. To elucidate the mechanism of hsa-miR-148a regulating osteoclastogenesis from CD14+ PBMCs.MethodsPredict the target gene of hsa-miR-148a by using various miRNA target prediction software tools such as TargetScan and PicTar. To create wide type (WT) luciferase reporter vector WT-pGL3-MAFB-3'UTR, a segment of the MAFB 3'UTR including the putative target site was PCR amplified and cloned into the downstream of the stop codon in the pGL3-Control Firefly Luciferase reporter vector. The QuickChange site-directed mutagenesis kit was used to introduce mutations into the target gene, resulting in mutant (MUT) luciferase reporter vector MUT-pGL3-MAFB-3'UTR. These two reporter vectors were cotransfected with pre-miR-148a or miR-C into PBMCs, and Dual Luciferase Reporter Assay System was used to detect the luminescent signal. To directly detect the pre-miR-148a suppression validity of the target gene, PBMCs were only transfected with pre-miR-148a. The protein and mRNA levels of target gene were measured by Western blotting and qRT-PCR. The segment of the MAFB CDS including the putative target site was PCR amplified and cloned into pcDNA3.1(+) vector, resulting in WT target gene expression vecor. The QuickChange site-directed mutagenesis kit was used to introduce mutations into the target gene, resulting in MUT target gene expression vector. We cotransfected the WT or MUT target gene expression vector, with pre-miR-148a or miR-C into rh-MCSF and hRANKL-induced PBMCs to determine TRAP mRNA levels by qRT-PCR, and the target gene protein expression level was detected by Western blotting analysis.Results(1) MAFB is the target gene of hsa-miR-148a predicted by miRNA target prediction software tools such as TargetScan and PicTar. (2) Compared with control, cotransfection of pre-miR-148a with WT-pGL3-MAFB-3'UTR significantly suppressed the luciferase activity. Cotransfection of pre-miR-148a with MUT-pGL3-MAFB-3'UTR abolished this repression, confirmed MAFB is the target gene of hsa-miR-148a. (3) Compared with control, transfection of pre-miR-148a downregulated endogenous MAFB protein levels. However, no changes in MAFB mRNA levels was detected. (4) The increased TRAP mRNA levels by pre-miR-148a was rescued by the mutant MAFB expression vector. Western blotting showed that the mutant MAFB CDS construct was also able to rescue the pre-miR-148a-induced downregulation of MAFB protein levels. The results suggested that hsa-miR-148a promotes osteoclast differentiation by targetting MAFB.Conclusion(1) We have constructed WT and MUT MAFB 3'UTR luciferase reporter vector, and WT and MUT MAFB expression vector. (2) The fact that MAFB is target gene of hsa-miR-148a, have been predicted by miRNA target prediction software tools and identified by Luciferase reporter vector measurement. (3) hsa-miR-148a promotes osteoclast differentiation by repressing MAFB expression at the post-transcriptional level. (4) MAFB is the most important target of hsa-miR-148a in osteoclast differentiation.