Study On The Dual Function Of MiR-338-3p In Mouse Odontoblast-osteoblast Differentiation

1. miR-338-3p promotes differentiation of mDPC6T into odontoblast-like cells by targeting Runx2Objectives:Odontoblast cells, which are derived from neural crest origin of dental papilla, are very special terminally differentiated cells. They are formed by the induction of certain signaling pathway. The differentiation of odontoblasts is regulated by several signaling pathway, signaling molecules, transcription factor and small RNAs. MicroRNAs, which are small non-coding RNAs (20-25nt), can play important roles in cell proliferation, differentiation and organ development. In this study, we try to explore the function and the exact mechanism of miR-338-3p during odontoblast differentiation.Methods:In order to get an in vitro odontoblast differentiation model, we cultured the mDPC6T cells in the induction medium. Real-time PCR was used to detect the expression pattern of miRNAs during odobtoblast differentiation. Then lentivirus was infected and inhibitors were transfected into mDPC6T to over-express and inhibit the expression of miR-338-3p, respectively. Then the infection and transfection cells were cultured in the induction medium for14days. Total RNA was extracted. Real-time PCR was performed to detect the expression of miR-338-3p and odontoblastic differentiation related makers (Dspp, Dmpl and Alp). Von Kossa staining was used to detect the formation of mineralization nodules. Bioinformatics analysis showed Runx2might be one of the target genes of miR-338-3p. After over-expression and inhibition of miR-338-3p, real-time PCR and western blot were used to detect the expression of Runx2at the mRNA and protein level. Dual luciferase analysis was performed of the relative luciferase activity.Results:miR-338-3p was significantly up-regulated during the differentiation of mDPC6T cells into odontoblast-like cells. Over-expression of miR-338-3p could promote the expression level of odontoblastic differentiation related makers (Dspp, Dmpl and Alp) and increased the formation of mineralization nodules. On the contrary, inhibition of miR-338-3p could impair odontoblast differentiation. Over-expression of miR-338-3p caused a decreased in the expression of Runx2at both mRNA and protein levels, while Runx2expression was increased after treatment with miR-338-3p inhibitors. Furthermore, the lucifarase activity was significantly suppressed by ectopic expression of miR-338-3p.Conclusion:miR-338-3p promotes odontoblast differentiation through targeting the3’-UTR of Runx2. 2. miRNA-338-3p regulates osteogenic differentiation of mouse bone marrow stromal stem cells by targeting Runx2and Fgfr2Objectives:Mouse bone marrow stromal stem cells (BMSCs) have been reported to be a population of self-renewing multipotent cells. They can differentiate into several lineages in response to stimulation by multiple environmental factors. BMSCs can induce into osteoblasts after culturing in osteoblast induction medium. Both odontoblast and osteoblast progenitor cells are derived from cranial neural crest, the differentiation process of these two types cell are similar. Our previously study indicated that miR-338-3p can promote odontoblast differentiation by targeting the3’-UTR of Runx2. As an important transcriptional factor, Runx2also can play an important role during osteoblast differentiation. But the function of miR-338-3p in osteoblast differentiation is still unclear. In this study, we try to detect the expression of miR-338-3p in osteoblast differentiation and the function in bone’metabolism related disease-osteoporosis.Methods:The primary BMSCs were cultured in osteoblast differentiation medium. Real-time PCR was used to detect the expression pattern of osteoblast differentiation markers (Runx2, Alp, Opn, Ocn and Coll). Then we performed Alizarin Red staining to confirm the successful established of this induction model. The expression of miR-338-3p during osteoblast differentiation was determined by qRT-PCR. At the same time, the primary BMSCs were infected with lentivirus to over-express and transfected with inhibitors to inhibit miR-338-3p for7days. The expression level miR-338-3p and osteoblast differentiation markers were analyzed by qRT-PCR. Bioinformatics analysis predicted that both Runx2and Fgfr2are the target genes of miR-3383-p. qRT-PCR and western blot were used to detect the expression of Runx2and Fgfr2. The regulatory relationship between miR-338-3p and these target genes was confirmed by a dual luciferase reporter assay. We established an osteoporosis mouse by performing ovariectomy (OVX), a model for early stage of primary osteoporosis. The H&E staining and micro-CT was showed the well-established of this osteoporosis model. qRT-PCR and western blot were applied to detect the expression of miR-338-3p and Runx2and Fgfr2. Then the miR-338-3p inhibitors were transfeted to the OVX and control primary BMSCs and cultured these cells in induction medium. The expression of the osteoblast differentiation markers were detected by qRT-PCR.Results:Both qRT-PCR and Alizarin Red staining verified that the osteoblast differentiation model was successfully established. miR-338-3p was significantly down-regulated during this process. The key osteogenic specific genes(Alp, Opn, Ocn, and Coll) were significantly down-regulated and the formation of mineralized nodules was decreased due to the over-expression of miR-338-3p. While, after knocking down of miR-338-3p could up-regulate the osteoblast differentiation markers. And there were more mineralized nodules formation. Over-expression and inhibition of miR-338-3p could down-regulate and up-regulate the expression of Runx2and Fgfr2. Dual luciferase assays also confirmed the regulatory role of miR-338-3p on Runx2and Fgfr2. As another transcription factor of osteoblast differentiation, the expression of Osx was also affected, but there were no direct binding sites for miR-338-3p in the3’-UTR of Osx. The H&E staining and micro-CT suggested the osteoporosis mouse model was successfully established. Compared with the sham group mice, miR-338-3p was up-regulated in the osteoporosis mice. The target genes Runx2and Fgfr2was down-regulated at both mRNA and protein level. The inhibition of miR-338-3p can partially rescue the cell lineage commitment disorder of BMSCs from osteoporosis bone marrow.Conclusion:miR-338-3p regulates osteogenesis differentiation of BMSCs through targeting Runx2and Fgfr2. miR-338-3p can play an important role in mice osteoporosis.