Function Expression Of MiR-345-3p In Osteogenic Differentiation Induced By Cycling Stretch Of Rat Bone Mesenchymal Stem Cells

BackgroundMechanical force induced alveolar bone reconstruction is one of the most important theoretical basis of tooth movement biology. The mechanism of mechanical force to activate cells of bone reconstruction not only is a research emphasis in the field of orthodontics, but also the oral and maxillofacial surgery, orthopedic areas. In vivo and vitro experiments and clinical applications can be found in the orthodontic tooth movement, tension, fracture repair process, such as the appropriate tension can stimulate and rebuild the growth of bone tissue. In the process of new bone formation, bone mesenchymal stem cells (BMSCs) under mechanical stretch tension can raise to the tension areas, proliferation, differentiation and development, which is the cytological basis of the formation of new bone, which is a key of maintaining the stability of the skeletal system. Microrna (miRNA) as a kind of 22-24 bp’s long noncoding single-stranded RNA, widely exists in various organisms, which regulates the expression of genes by inhibiting cell protein levels. The inhibition of mRNA translation by miRNAs has been found to be an important signaling pathway that regulates bone formation. Recent years, more and more researches have shown that miRNA is closely related to osteogenic differentiation of bone mesenchymal stem cells. However there were rare system and in-depth researches in the past about the miRNA involved in stretch-induced osteogenic differentiation of BMSCs.In our early experiments, we detected the miRNA’s gene expression of rat BMSCs of 12 hours and 0 hour stimulated by cyclic mechanical stretch via the gene microarray detection technology, and analyzed and selected a series of miRNAs of discrepant expression of before and after cyclic mechanical stretch. We selected the miR-345-3p whose expression significantly decreased, to verify its biological role in stretch-induced osteogenic differentiation, and to predict its target genes by bioinformatics technology.ObjectiveThe aim of this experiment was to investigate and verify biological effects of miR-345-3p in stretch-induced osteogenic differentiation, and to predict its target genes by bioinformatics technology. Explore the mechanisms of stretch-induced osteogenic differentiation of rat BMSCs preliminary from the angle of miRNA to provide theoretical and experimental basis for orthodontic bone reconstruction and distraction osteogenesis and new strategy of diagnosis and treatment of oral diseases under mechanical force.Method1.Identification of isolated rat bone mesenchymal stem cells:The rat BMSCs were isolated by the whole marrow-adherence way, amplification in vitro, purification to obtain high purity BMSCs of rats. The morphology of cells was observed with light microscope. The proliferation ability was tested by CCK-8 and alizarin red staining, ALP stain, oil red O staining were used to detect osteogenic differentiation and adipogenic differentiation. Flow cytometry was used to identify cell surface antigen.2.Observation ALP activity, the expression of osteogenic genes and miR-345-3p over time after application of the cyclic stretch on rat BMSCs:In this experiment, cells were respectively divided into 4 groups according to duration of Oh,3h,6h, and 12h, and then they were applied by cyclic stretch of 10%, 1Hz. ALP activity and the expressions of Runx2、Osterix and miR-345-3p over time were tested by RT-PCR.3.Observation the role of miR-345-3p in cyclic stretch-induced osteogenic differentiation:miR-345-3p was both overexpressed and inhibited in rat BMSCs by transfection technology. After application of 10%, 1Hz cyclic stretch for 3h, osteogenic genes were measured by RT-PCR and Western blot to figure out the role of miR-345-3p in cyclic stretch-induced osteogenic differentiation.4. Select the candidate target genes of miR-345-3p via bioinformatics websites and softwares.Results1.BMSCs which were isolated from bone marrow were detected by phenotypes and flow cytometer, and the results showed CD44, CD90 surface antigens were positive, and CD34, CD45 surface antigens of culture cells were negative, and BMSCs could differentiate into osteocytes and adipocytes. All basic features indicated that these cells were rat BMSCs.2.After cyclic stretch (10% of the tensile strength and frequency of 1 Hz) on rat BMSCs, there was a time-dependent increase in the expression of ALP activity and immediately after loading (P<0.05); the mRNA expression levels of Runx2 and Osterix gradually increased and respectively after 3 hours and 6 hours (P<0.05); the expression of miR-345-3p gradually reduced, and reached to trough in 3 hours (P<0.01), then gradually raised, but still below the level of the control group.3.Increased the expression of miR-345-3P could significantly reduce the cyclic stretch-induced osteogenic differentiation in rat BMSCs. RT-PCR results showed that the expressions of ALP, Runx2, Osterix reduced (P<0.05) in the miR-345-3P mimic group than in the control group, and Western blot showed that the expressions of ALP and Runx2 decreased significantly in the miR-345-3P mimic group than in the control group. Reduced the expression of miR-345-3P could increase the cyclic stretch-induced osteogenic differentiation. RT-PCR results showed that the expressions of ALP, Runx2, Osterix enhanced in the miR-345-3P inhibitor group than in the control group (P<0.05), and Western blot results showed that the expressions of ALP and Runx2 significantly increased in the miR-345-3P inhibitor group than in the control group.4.Hoxa2, IGF2, CBFB, BCL2, AXIN2, FN1, et al, might be the candidate target genes of miR-345-3p by bioinformatics prediction.Conclusions1.The pure rat BMSCs could be obtained by using the whole marrow-adherence way and they can be induced into the osteocytic and adipogenic lineages.2.Osteogenic differentiation could be induced by cyclic stretch (10% of the tensile strength and frequency of 1 Hz) of rat BMSCs in vitro; miR-345-3p could respond to mechanical stimulation, which confirmed that miR-345-3p was involved in cyclic stretch-induced osteogenic differentiation in rat BMSCs.3.The miR-345-3p played a negative role in regulation cyclic stretch-induced osteogenic differentiation in rat BMSCs.4.This research helps to better understand the mechanism of orthodontic bone reconstruction and distraction osteogenesis, and provide theoretical basis for guiding clinical practice.