MicroRNA Profiling During Osteoblast Mineralization And The Functional Study Of MiR-93

Part one Study on the microRNA profile during the mineralization of mouse primary osteoblastsObjectiveTo establish the mineralization cell model of osteoblasts derived from calvaria of C57BL/6mice. To study the microRNA (miRNA) expression profile during the differentiation and mineralization of osteoblasts.MethodsFirstly, primary osteoblasts were separated from the calvaria of C57BL/6mice by the method of surgery. Osteoblasts were purified by the method of adherent separation. Secondly, the isolated osteoblasts were differentiated and mineralized in the presence of50mg/L ascorbic acid and lOmM β-glycerophosphate ((3-GP) for21days in a humidified5%CO2atmosphere at37℃. Thirdly, the mineralization of osteoblasts was assessed by Alizarin Red S staining. Osteoblasts were stained to observe mineralized matrix formation in osteoblasts after cultured for14and21days in differentiation medium. The osteoblast mineralization model derived from mouse calvaria primary osteoblasts was established. Lastly, the differentiated miRNA expression profile during the differentiation and mineralization of osteoblasts was screened by miRNA microarray and verified by quantitative real-time PCR (qRT-PCR).Results(1) Primary ostteoblasts were successfully separated and purified from the calvaria of C57BL/6mice by means of surgery and the technique of differential anchoring velocity.(2) After14days of culture of the osteoblasts, mineralized nodules appeared, and mineralized nodules increased gradually. The mineralized nodules obviously increased on day21compaered with that of day14.(3) In the present study, miRNA microarray showed the miRNAs which were differently expressed during the differentiation and mineralization of osteoblasts. mmu-miR-98, mmu-miR-130a, mmu-miR-210were up-regulated obviously and mmu-miR-93, mmu-miR-103, mmu-miR-92b, mmu-miR-674, mmu-miR-351, mmu-miR-24were down-regulated apparently.(4) The results of qRT-PCR were consistent with that of miRNA microarray, which showed accuracy of qRT-PCR for verification of differented miRNA expression.ConclusionThe osteoblast mineralization model deriving from the calvaria of C57BL/6mice was established successfully. miRNA microarray showed the miRNAs which were differently expressed during the mineralization of primary osteoblasts. mmu-miR-98, mmu-miR-130a, mmu-miR-210were up-regulated, reversely, mmu-miR-93, mmu-miR-103, mmu-miR-92b, mmu-miR-674, mmu-miR-351and mmu-miR-24were down-regulated apparently. mmu-miR-93was the most dramatically downregulated miRNA. Part Two Study on the effect of miR-93in osteoblast mineralizationObjectiveTo investigate the function of miR-93in the mineralization of primary osteoblasts.MethodsThe miR-93expression profile was detected by qRT-PCR during osteoblasts mineralization induced by ascorbic acid and (β-GP. We designed primers according to the precursor sequence of miR-93, and constructed the expression vector of miR-93by using the pAJ vector. The expression vector was named "pre-miR-93". After the transfection of primary osteoblasts with pre-miR-93, osteoblast mineralization was induced by the addition of ascorbic acid and P-GP. quantitative real-time PCR (qRT-PCR) analysis detected the expression profile of miR-93. The growth and function of osteoblasts were assessed by Alizarin Red S staining. The Sp7mRNA and protein levels were detected by qRT-PCR and Western blot analysis.Results(1) The expression of miR-93in primary osteoblasts was detected after treatment with ascorbic acid and β-glycerophosphate and decreased progressively with time.(2) High expression of miR-93was obtained in primary osteoblasts after transfection with the expression vector of miR-93constructed by using pAJ-CMV-eGFP vector.(3) Compared with control cells, miR-93overexpression inhibites the formation of mineralized nodules. Transfection of pre-miR-93reduced levels of Sp7protein levels without affecting its mRNA levels. ConclusionThe expression vector of miR-93was constructed. miR-93overexpression inhibited osteoblast mineralization. Part Three The roles and mechanisms of miR-93/Sp7loop in osteoblast mineralizationObjectiveTo predict and verify the interaction of miR-93and its target gene. To elucidate the roles of miR-93/Sp7loop in osteoblast mineralization.MethodsVarious miRNA target prediction software tools such as TargetScan and Rna22were used to predict the target genes of miR-93. To create wide type (WT) luciferase reporter vector WT-pGL3-Sp7-CDS, a segment of the Sp7CDS 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-Sp7-CDS. These two reporter vectors were cotransfected with pre-miR-93or miR-C into primary osteoblasts, and Dual Luciferase Reporter Assay System was used to detect the luminescent signal. To directly detect the pre-miR-93suppression validity of the target gene, osteoblasts were transfected with pre-miR-93. The protein and mRNA levels of target gene were measured by Western blot and qRT-PCR. The segment of the Sp7CDS including the putative target site was PCR amplified and cloned into pAJ 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-93or miR-Control into osteoblasts for21days, and determined the target gene protein expression levels and the mineralization levels. The putative transcription factors of mmu-miR-93were found by TF search. Electrophoretic mobility shift assay EMSA and Chromatin Immunoprecipitation (CHIP) were used to verify the combination of Sp7and the promoter of miR-93. To create wide type (WT) miR-93promoter luciferase reporter vector WT-pGL3-promoter, a segment of the miR-93promoter including the putative target site was PCR amplified and cloned into the downstream of the stop codon in the pGL3-basic 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-promoter. These two reporter vectors were cotransfected with pre-miR-93or miR-C into primary osteoblasts, and Dual Luciferase Reporter Assay System was used to detect the luminescent signal. The full length of the Sp7was PCR amplified and cloned into pAJ vector, resulting in Sp7expression vecor. To directly detect the Sp7suppression validity of miR-93, osteoblasts were only transfected with Sp7expression plasmid. We transducted the Sp7expression vector and determined the miR-93levels by qRT-PCR. The sh-Sp7was cloned into pAJ vector, resulting in sh-Sp7expression vecor. We transducted the sh-Sp7expression vector and determined the miR-93levels by qRT-PCR.Results(1) Sp7was the target gene of miR-93predicted by miRNA target prediction software tools such as Rna22.(2) Compared with the control, cotransfection of pre-miR-93with WT-pGL3-Sp7-CDS significantly suppressed the luciferase activity. Cotransfection of pre-miR-93with MUT-pGL3-Sp7-CDS abolished this repression, confirmed Sp7is the target gene of miR-93.(3) Compared with the control, transfection of pre-miR-93downregulated endogenous Sp7protein levels. However, no change in Sp7mRNA levels was detected. The results suggested that miR-93inhibit osteoblast mineralization by targetting Sp7.(4) Mutant Sp7CDS rescued the inhibition of miR-93on osteoblast mineralization and the protein level of Sp7.(5) miR-93transcription start site (TSS) was located on mouse chromosome5(Genbank:NC₀00071.5). Sp7was predicted to be the transcription factor of miR-93by TF search. A potential binding site for Sp7resided just upstream of the miR-93TSS.(6) EMSA experiment showed that Sp7combined with miR-93promoter.(7) CHIP experiment revealed that Sp7bound to the miR-93promoter through the putative binding sites.(8) Compared with the control, miR-93promoter activities were significantly reduced by Sp7. In contrast, there was no significant difference in the promoter activities between the Sp7binding site mutation-transfected and control cells. These data suggested that Sp7directly repressed transcription via physical binding resulting in decreased expression of miR-93.(9) Compared with the control, transfection of Sp7downregulated endogenous miR-93expression levels. While, transfection of sh-Sp7upregulated endogenous miR-93expression level. The results suggested that Sp7inhibit miR-93expression to promote osteoblast mineralization.Conclusion(1) We constructed WT and MUT Sp7CDS luciferase reporter vector, and WT and MUT miR-93promoter vector.(2) The fact that Sp7was target gene of miR-93and also was a transcription factor of miR-93was predicted by miRNA target prediction software or TF search tools and identified by Luciferase reporter vector, EMSA, and CHIP expriments. (3) miR-93inhibited osteoblast mineralization by repressing Sp7expression at the post-transcriptional level. Sp7reduced miR-93expression as a transcription factor of miR-93.(4) Sp7was the most important target of miR-93in osteoblast mineralization. Sp7directly repressed transcription via physical binding, and resulted in down-regulated expression of miR-93. They formed a unique autoregulatory feedback loop in regulating osteoblast mineralization.