MicroRNAs Regulate Satb2-induced Osteogenic Differentiation In Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) are a potential cell source capable of differentiating into several mesenchymal cell types including osteoblasts and chondrocytes. Maximizing the capacity of BMSCs to regenerate bone requires methods for enhancing their osteogenic potential. The combination of gene therapies and stem cells could be an optimal clinical strategy for tissue repair. A member of the special family of AT-rich binding transcription factors, special AT-rich sequence-binding protein 2 (Satb2) was demonstrated to play a pivotal role in craniofacial patterning and osteoblast differentiation, indicating that Satb2 can serve as a potent osteo-inductive factor in bone regeneration. However, systemic research on the mechanism of Satb2-induced osteogenic differentiation is still obscure.In this study, we used lentiviral-mediated gene transfer of Satb2 into mouse BMSCs and investigated the capacity of Satb2 overexpression to promote osteogenic differentiation in vitro and in vivo.Then, we studied the differentially expressed miRNAs by Satb2 overexpression in mouse BMSCs using miRNA microarray. The target genes of the selected differentially expressed miRNAs were also analyzed in the gene ontology (GO) and KEGG biological pathway. This study may elucidate the mechanism of Satb2-induced osteogenic differentiation, and thus provide basis for tissue-engineered regeneration of bone around implants.Materials and methods:Part 1:The Satb2 coding sequence was amplified from mouse genomic DNA and was cloned into the pTA2 vector and transferred to generate the recombinant lentiviral expression vector LV-Satb2 using in vitro recombination.Then, we used lentiviral-mediated gene transfer of Satb2 into mouse BMSCs and investigated the capacity of Satb2 overexpression to promote osteogenic differentiation in vitro by Alkaline phosphatase (ALP) activity, ALP staining and Alizarin Red S staining. The expression of osteoblast-specific genes such as Runx2, Sp7, Atf4, and Bsp were examined by western bloting and Quantitative real time PCR (qRT-PCR) analysis.The β-tricalcium phosphate (β-TCP) scaffolds were cultured with BMSCs in osteogenic mediumfor 14 d. Then, the composition was placed into the thigh muscle pouch in both of the bilateral hind legs in C57BL6 mice. All animals were sacrificed 4 weeks post-surgery and the implants were harvested, then a half of the specimens were used for qRT-PCR analysis and the other half were subjected to bistological analyses.Part 2:The differentially expressed miRNAs by Satb2 overexpression in mouse BMSCs were detected by miRNA microarray. Genes with changes in expression levels of≥1.5-fold than those of the controls were defined as differentially expressed genes. These genes were subjected to GO term analysis and grouped into hierarchical categories to uncover the miRNA-gene regulatory network based on biological processes and molecular functions. Meanwhile, KEGG pathway annotation was also performed for these genes using the DAVID gene annotation tool.We selected miR-27a for target verification and functional analysis. To examine the effect of miR-27a on differentiation, BMSCs transfected with miR-27a and the negative control were cultured in osteogenic differentiation medium. ALP staining and Alizarin Red S staining were performed at 7 days and 14 days after inoculation, respectively. miR-27a was predicted to target BMP2, BMPRla, and Smad9, which are involved in TGF-β/BMP signaling pathway. For target verification, we used western blot to test the protein levels of BMP2, BMPR1 A, and Smad9.Part3:To further investigate miRNA-mRNA interaction network,, we studied the expression pattern of Sp7 and miR-27a during Satb2-induced osteogenic differentiation by western blotting and qRT-PCR. The expression of Sp7 was also examined by western blot and qRT-PCR in cells transduced with miR-27a.The expression of osteogenic factor such as Satb2, Sp7, ALP, and OCN were detected by qRT-PCR at 7 day’s osteogenic differentiation.Sp7 mRNA 3’UTRs containing the miR-27a-binding sequences for the mouse Sp7 gene were amplified by PCR from mouse genomic DNA. The PCR production were digested with XhoI and NotI, and then inserted into the psiCHECK-2 reporter vector downstream of the Renilla luciferase gene. Binding-region mutations were achieved using a Site-Directed Mutagenesis Kit. For luciferase assays, psiCHECK-2-SP7 3’UTR and pcDNA3.1-miR-27a were transiently transfected into 293T cells. After 24h transfection, firefly and Renilla luciferase activities were determined.Results:Parti:In this study, we first used lentiviral-mediated gene transfer of Satb2 into mouse BMSCs. We found that LV-Satb2 transduced BMSCs produced Satb2 protein and underwent rapid and marked osteogenic differentiation as demonstrated by increased osteoblastic gene expression including Runx2, Sp7, Atf4, and Bsp, ALP activity and Alizarin Red S staining. To analyze the induction of bone formation in vivo, LV-Satb2 transduced BMSCs were implanted into the hindlimbs of syngeneic mice, with β-tricalcium phosphate as the scaffolding material. Four weeks after implantation, transduction with LV-Satb2 greatly enhanced formation of new bone.Part2:Twenty-eight miRNAs were found to be differentially expressed. Of these, four under-expressed (miR-27a, miR-125a-5p, miR-466f-3p and miR-146b) and three novel over-expressed miRNAs (miR-17, miR-20a and miR-210) were selected and their expression were confirmed by qRT-PCR. The differentially expressed miRNAs were further subjected to GO and KEGG analysis. The highly enriched GOs and KEGG pathway showed target genes of these miRNAs were significantly involved in multiple biological processes (mesenchymal cell differentiation, bone formation, and skeletal development), and several osteogenic pathways (TGF-β/BMP, MAPK, and Wnt signaling pathway). Finally, miR-27a was selected for target verification and function analysis. BMP2, BMPR1A, and Smad9, members of the TGF-β/BMP superfamily, which were predicted to be target genes of miR-27a, were confirmed to be significantly up-regulated in Satb2-overexpressing cells by qRT-PCR. Overexpression of miR-27a significantly inhibited osetogenesis and repressed BMP2, BMPR1 A, and Smad9 expression.Part3:Western blot analysis revealed that expression of Sp7 was increased during the early stage of Satb2-induced osteogenic differentiation and it reached the peak at day 7, while the expression level of miR-27a was at the lowest at day 7. These data implied a reciprocal expression pattern between Sp7 and miR-27a in the course of Satb2-induced osteogenic differentiation. Both western blot and qRT-PCR analysis exhibited obviously decreased level of Sp7 in cells tranduced with miR-27a.Being assessed by the dual luciferase assay, significant decrease in relative luciferase activity was noted when psiCHECK-2-Sp7 3’UTR was cotransfected with miR-27a. This suppressive effect was abolished by site-directed mutagenesis of the seed region in the 3’UTR of Sp7 mRNA. These results confirmed that miR-27a could regulate Sp7 expression through a partially complementary binding site in the 3’UTR of Sp7.Conclusions:1. Lentiviral-mediated Satb2 overexpression can promote the osteogenic differentiation of BMSCs in vitro and to enhance bone formation through a tissue engineering technique that will be highly useful in bone regenerative medicine.2. We identified a number of the differentially regulated miRNAs, whose target genes involved in the TGF-β/BMP signaling pathway, play an important role in the early stage of Satb2-induced osteogenic differentiation.3. miR-27a may regulate Sp7 expression through direct targeting the 3’UTR of Sp7. Satb2 may work along with miR-27a and Sp7, constituting a regulatory feed-forward loop during osetogenic differentiation of BMSCs.