| Background:Bone mesenchymal stem cells (BMSCs), isolated from the bone marrow, have the potential to differentiate into multiple tissue cells types including osteoblasts, chondrocytes and adipocytes. The potential to differentiate into osteoblasts makes them attractive targets for applying in therapeutic applications on bone repair and tissue engineering. How to enhance the osteogenic differentiation of BMSCs has gained increasing attention in recent years.Special AT-rich sequence-binding protein 2 (SATB2) is a cloned member of the family of special AT-rich sequence-binding proteins that binds to nuclear matrix-attachment regions (MARs). SATB2 can bind to and regulate bone sialoprotein (BSP) and osteocalcin (OCN) gene, enhance the activity of both Runt-related transcription factor 2 (Runx2) and activate transcription factor 4 (ATF4), which suggests that SATB2 plays a critical role in osteoblast differentiation. Recently, SATB2 has been identified as a novel marker of osteoblastic differentiation. SATB2 can act not only as an activating or repressing DNA bound protein but also as a protein scaffold that enhances the activity of other DNA binding proteins. Therefore our research aims at the detailed mechanism of SATB2 during osteoblast differentiation.MicroRNAs (miRNA) are small RNA molecules that bind to the non-coding region of mRNA and regulate mRNA activity by inducing mRNA degradation or suppressing mRNA activity. MicroRNAs are involved in many progresses, including cell proliferation, apoptosis and autophagy. To date, studies have substantiated that miRNAs, such as mir-31,-34c,-204,-338-3p and so on, are involved in regulating the differentiation of BMSCs. Mir-205, known as a tumor suppressor, plays an important role in tumor cell proliferation and migration. Recent studies showed that mir-205 was down-regulated during the induction of osteogenic differentiation. However, the detailed mechanism remains unclear. What’s more, the role of mir-205 in BMSCs has not been characterized.Mir-205 inhibitor and mir-205 mimics were used to detect effects of mir-205 on the differentiation of BMSCs. And bioinformatics analysis and plasmid construction were used to illustrate the influence of mir-205 on STAB2 and Runx2. Our data showed that mir-205 negatively regulated osteogenic function in BMSCs by mediating STAB2 and Runx2 expression via ERK and p38 MAPK pathway.Aim:Through the primitive culture between bone marrow mesenchymal stem cells in vitro, detection of mir-205 change in the process of cell proliferation and cell differentiation. Inhibited by mir-205 inhibitor and simulation of mir-205 in the bone marrow mesenchymal stem cells express, observe change of mir-205, between bone marrow mesenchymal stem cells in the process of change. Observation Runx2 and the expression of SATB2 change at the same time, reveals the relationship between mir-205 and SATB2 and Runx2, to clarify mirnas adjust filling between bone marrow stem cell function provides strong evidence and new ideas.Methods:1. Mir-205 in the osteogenetic differentiation of mesenchymal stem cells in the bone marrow. Using bone marrow mesenchymal stem cells between the original generation methods to observe the role of mir-205 in the osteogenetic differentiation. Using RT-PCR test mi-205 in the osteogenetic differentiation. By joining mir-205 inhibitor and simulation to change things between bone marrow mesenchymal stem cells in the expression of mir-205, by BSP and protein expression of OPN changing to reflect the differentiation between bone marrow mesenchymal stem cells.2. Mir-205 of SATB2 and Runx2 adjustment. We first through bioinformatics analysis, found that mir-205 and SATB2 3 ’UTR region exists complementary base pairing, suggests there may be a close relationship between. By Western blot experiments testing found that high expression can reduce the protein expression of SATB2 mir-205. By RT-PCR experiments to detect Runx2 is a target mRNA mir-205.3. SATB2 regulating mir-205 mediated between bone marrow osteogenesis of mesenchymal stem cell differentiation. We first construct PEGFP-N1 plasmid, make SATB2 expressed in bone marrow mesenchymal stem cells between higher. By Western blot experiments testing Runx2 protein changes. We through ALP and OCN detecting SATB2 whether can effectively increase bone marrow between the osteogenetic differentiation capacity of mesenchymal stem cells.4. MAPK signaling pathways in mir-205 to adjust the role of bone marrow mesenchymal stem cells between. By Western blot detection MAPK signaling pathways phosphorylation level of change. Observation of MAPK signal pathway after intervention mir-205 change.Results:1. Mir-205 is down-regulated during the process of osteogenic differentiation.Quantitative real-time PCR results showed that mir-205 expression was reduced in a time-dependent manner during the process of BMSCs osteogenic differentiation. The mir-205 level in BMSCs decreased after treatment with DM for 12h and the mir-205 level reduced further with the extension of time. To further conform the mir-205 expression, northern blot analysis was used and results were in good consistence with qRT-PCR. We also detected the mir-205 level in BMSCs culture with GM, and no significant change was found. These data suggested that mir-205 was down-regulated during the process of osteogenic differentiation in BMSCs.2. Inhibition of mir-205 enhances osteogenic differentiation.To investigate the role of mir-205 in the osteogenic differentiation in BMSCs, we used mir-205 inhibitor or mir-205 mimics to change the expression of mir-205. Protein expression of bone sialoprotein (BSP), osteopontin (OPN) were examined in BMSCs cultured with DM after transfection for 48h. Western blot analysis data showed that BSP and OPN protein levels were significantly decreased in BMSCs in mir-205 mimic group. Inhibition of mir-205 increased protein expression of BSP and OPN. What’s more, ALP activity and OCN secretion levels were increased in mir-205 down-regulation BMSC cells, and decreased in mir-205 over-expression BMSC cells. Inhibition of mir-205 dramatically increased the mineralization effects compared to NC group, but over-expression of mir-205 diminished the mineralization effects. Our results suggested that mir-205 negatively regulated the osteogenic function in the differentiation of BMSCs.3. SATB2 and Runx2 expression are regulated by mir-205 in BMSCs.SATB2 is a key transcription factor in regulating osteogenic function in BMSCs, and bioinformatics analysis showed that mir-205 was predicted as a potential miRNA binding motifs on the SATB2 3’UTR. To confirm the combination between SATB2 and mir-205, BMSCs were transfected with mir-205 mimics and luciferase reporter. Next, we investigated the effect of mir-205 on the SATB2 expression in BMSCs. mir-205 inhibitor or mir-205 mimics were transfected into BMSCs culture for 48h, and western blot results showed that the protein level of SATB2 was down-regulated in mir-205 mimics group and the protein expression of STAB2 was reduced. As expected, the SATB2 protein level was up-regulated in mir-205 inhibitor group. Runx2 has been reported as an early key transcription factor regulating osteogenesis, and has been identified as a direct target of mir-205. Our results suggested that mir-205 was involved in the regulation of SATB2 and Runx2 expression.4. SATB2 regulates mir-205-mediated osteoblastic differentiation in BMSCs.Next, we tried to investigate whether SATB2 could regulate mir-205-mediated osteogenic differentiation. PEGFP-N1 plasmids were used to construct STAB2 overexpression vectors, and western blot data showed that SATB2 protein expression was significantly up-regulated in BMSCs and mir-205-overexpressing BMSCs (Fig.4A and 4B). Previous studies showed that SATB2 could enhance the activity of Runx2, so we also detected the expression of Runx2 in SATB2-overexpressing BMSCs. Our results indicated that up-regulation of SATB2 increased compared to NC. Then, we detected the ALP activity and OCN levels, and as expected, overexpression of SATB2 increased ALP activity and OCN levels. Our data indicated that SATB2 could regulate mir-205-mediated osteoblastic differentiation in BMSCs.5. Inhibition of mir-205 increases the phosphorylation of ERK and p38 MAPK in BMSCs.Extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) signaling pathways have been reported to be mediated in the osteogenic differentiation of BMSCs. Therefore, we investigated whether ERK/p38 MAPK pathways were involved in mir-205 mediated osteogenic differentiation in BMSCs. BMSCs were cultured in DM and phosphorylation of ERK and p38 increased in a time-dependent way during the process of osteogenic differentiation. After transfected with NC, mir-205 inhibitor or mimics, we found that inhibition of mir-205 increased phosphorylation of ERK and p38 MAPK, while overexpression of mir-205 inhibited activity of ERK and p38 MAPK. Our results indicated that mir-205 negatively regulated osteogenic differentiation via ERK/MPAK pathway.Conclusions:In our experiment, we through the primitive culture between bone marrow mesenchymal stem cells in vitro detection of mir-205 change in the process of cell proliferation and cell differentiation, after by mir-205 inhibitor and mimetic change the expression of mir-205, to observe the change after mi-205, the change in the process of cell differentiation. Through testing Runx2 and SATB2 expression change, observe the relationship between mir-205 and SATB2 and Runx2. In addition. We by detecting differentiation ERK and P38MAPK pathway related in the process of change, found that mir-205 May be affected by adjusting the MAPK pathway between bone marrow mesenchymal stem cell differentiation. Our research to understand the charge between miRNA regulate bone marrow stem cells function provides strong evidence and new ideas. |
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