Abstract
Osteogenic differentiation is an important process of new bone formation, microRNA-409-3p (miR-409-3p) has been reported to be up-regulated in the osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The present study aimed to investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism. The expression of miR-409-3p in osteoblast (human skull osteoblast, HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to suppressor of cancer cell invasion (SCAI) in MSC-B was investigated by performing a dual-luciferase reporter gene assay. MSC-B was selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase (ALP) activity, Alizarin Red staining, and the expression of osteogenic markers (ALP, osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2)) in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. Additionally, the Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 (DKK-1) to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs and showed an increasing time-dependent trend on the 0 and 21st day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI up-regulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes (Axis inhibition protein 1 (AXIN1), β-catenin, Lymphoid Enhancer Binding Factor 1, Cellular-myelocytomatosis (c-myc) and cyclin D1). Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by down-regulating SCAI expression.
Highlights
Mesenchymal stem cells (MSCs) extracted from bone marrow can form osteoblasts through osteogenic differentiation, which was first discovered by Friedenstein et al in the late 1960s to early 1970s [1]
After human skull osteoblast (HCO) and MSCs cells were cultured to a degree of fusion of 80–90%, we detected the expression of miR-409-3p in HCO, MSC-A, MSC-B, and MSC-U, and found that miR-409-3p expression of MSCs were lower than HCO (Figure 1A, P
MSCs were induced by the osteogenic induction medium, and the results showed that the alkaline phosphatase (ALP) activity of MSCs continues to increase with the osteogenic differentiation of HCO and MSCs on days 0, 7, 21 (Figure 1B, P
Summary
Mesenchymal stem cells (MSCs) extracted from bone marrow can form osteoblasts through osteogenic differentiation, which was first discovered by Friedenstein et al in the late 1960s to early 1970s [1]. Osteoblasts participate in the formation of bone and work together with osteoclasts, which is crucial for the repair of fractures, bone development and maintenance [2] MicroRNAs (miRNAs) are small and single-stranded RNAs that play important regulatory roles in mammals by pairing with mRNAs of target genes to guide their post-transcriptional inhibition [5], which has no effect on the stability of target mRNA. Their target genes play crucial roles in controlling the migration, differentiation, apoptosis and cell cycle progression of cancer-related cells. Our research aims to get the role of miR-409-3p on osteoblastic differentiation and the underlying mechanism of this process
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