MicroRNA Profiling During Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Subjected To Cyclical Stress And Function Study Of MiR-503-5p

BackgroundClinically, orthodontic forces induce remodeling of local alveolar bone, causing bone resorption and formation, ultimately resulting in tooth movement. As important regulator of bone metabolism, mechanical stimulation plays a crucial role in bone steady equilibrium. A variety of cells are involved in the local bone remodeling process. Among these, the active osteoblasts are indispensible sensor cells on the bone surface for reception of mechanical loads, and also act as the effector cells that increase bone formation products. After the development of individual body, osteoblasts do not have the proliferation ability and mainly derive from the differentiation of pluripotent stem cells including BMSCs. As one of the most commonly used cells in tissue engineering, BMSCs have the strong potency of self-renewal and multi-directional differentiation, and is also one of the mechano-sensitive cells. Mechanical force can change its functional status and induce it differentiate into osteocytes and bone lining cells. Researches show that activation of BMSCs caused by tensile stretch is a key factor in local alveolar bone reconstruction process. For the past few years, as the increasingly deepening of research about mechanical stimulation and BMSCs osteogenic differentiation, the molecular regulatory mechanism underlying has become a new research hotspot.MicroRNA is a class of small molecular non-protein coding RNA that containing an average of 22-25bp and existing in eukaryotic organisms. Through specific pairing with target mRNA, it could give rise to the degradation or repressed translation of it, consequently regulating the target gene at the post-transcriptional level. MiRNAs play important roles in different kinds of regulation processes including tumor biological behavior, stem cell proliferation, pluripotent maintenance and differentiation. With the deepening of the research, a variety of miRNAs have been proved to participate in the regulation process of osteogenic differentiation of BMSCs. The study has found that the miRNA-335-5p can directly regulate the Wnt pathway inhibitor DKK1 to promote the osteogenic differentiation of a variety of adult stem cells such as C3H10T-1/2、 MC3T3-E1、MLO-A5 and MLO-Y4. MiR-29b activates osteogenesis differentiation by down-regulating inhibiting factor of osteogenic differentiation, such as HDAC4, TGF3, and ACVR2A. However, in the process of stretch mediated osteogenesis, the role and regulatory mechanism of miRNA in the osteogenic differentiation of BMSCs remains to be further investigated. Therefore, our experiment aims to search for the mechano-sensitive miRNAs through the miRNA array and conduct a preliminary study on its effect and mechanism in the BMSCs osteogenic differentiation.ObjectiveFrom the viewpoint of cell and molecular biology, to explore the microRNA profile changes during osteogenic differentiation of bone marrow mesenchymal stem cells subjected to cyclical stretch and further verify the regulatory function of miR-503-5p. Then to elucidate the local bone reconstruction mechanism during clinical orthodontic tooth movement.Methods(1) The vitro isolation, cultivation and identification of rBMSCs.(2) Establishment of mechanical loading system in vitro and then select the optimum condition for cyclical stretch induced rBMSCs osteogenic differentiation.(3) Cyclical stretch was applied for rBMSCs using the optimum condition to screen the significantly expressed miRNA by utilizing the miRNA array. Then verify the results by qRT-PCR. Finally, we select the targeted miR-503-5p in combination with related literature.(4) To explore expression pattern of miR-503-5p in the osteogenic differentiation process and justify its regulation function by transient transfection with miRNA-503 mimics/inhibitor and detection of osteogenesis related factors(ALP、Runx2)expression changes in mRNA and protein level.Results(1) The success of vitro isolation and cultivation of rBMSCs. Flow Cytometry analysis revealed that the cells surface markers for rBMSCs is CD44/CD90 (+) and CD34/45(-). After osteogenic induction for 28 days, calcium salt deposition nodules can be observed after Alizarin red staining while after adipogenesis induction for 28 days, lipid droplets formed after Oil red O staining.(2) The success of establishment of mechanical loading system, and the ALP activity assay revealed that the optimum condition for BMSCs osteogenic differentiation is:1Hz,10% elongation and 12h.(3) The results of miRNA array reveal that in all three samples, compared with the control group, nine miRNAs expressed differently, two upregulated and seven downregulated. qRT-PCR verification results were consistent with the results of the microarray.(4) MiR-503-5p expression during cyclical stretch induced osteogenic differentiation process was time-dependent and reached its minimum level at 12h after stimulus loaded (p<0.01). The RNA and protein expression levels of Runx2, ALP in the miR-503-5p mimics group were lower than those in the control group, while those were higher in the miR-503-5p inhibitor group after transient transfection with miR-503-5p inhibitor.Conclusion(1) The expression of nine miRNAs have significant differences during the osteogenic differentiation of BMSCs subjected to cyclical stretch through microarray, among these miR-503-5p is the most notable.(2) MiR-503-5p inhibits osteogenic differentiation of rBMSCs subjected to cyclical stretch.