Effect Of MiR-132-3p On Proliferation And Osteogenic Differentiation Of MC3T3-E1 Cells By Targeting Smad5 Under Cyclic Tensile Stress

Objective:The reconstruction of periodontal tissue under stress is the biological basis of clinical malocclusion.Under mechanical stress,the process of cell osteogenic differentiation is regulated by a variety of signaling pathways.At the same time,more and more studies have found that Micro RNA(miRNA)is one of the important regular factors in osteogenic differentiation.However,the role of miRNAs in the process of TGF-?/BMP signaling pathway or its related signaling molecules regulating cell osteogenic differentiation has not been reported.Therefore,this study intends to screen the differential expression of miRNAs in MC3T3-E1 cells under 12% cyclic tensile stress and identify differential miRNAs which related to TGF-?/BMP signaling pathway.Then,the target genes of miRNAs in the TGF-?/BMP signaling pathway and their effects on cell osteogenic proliferation and differentiation were further determined.To provide a new idea for clarifying the mechanism of local bone remodeling during orthodontic treatment.Methods:1.Using miRNA gene chip technology to screen out differentially expressed miRNAs in MC3T3-E1 cells under 12% stretch stress,and combined with literature reports to verify the miRNAs that associated with TGF-?/BMP signaling pathway by qRT-PCR.2.Based on the results of Method 1,the differentially expressed miRNA was determined.Bioinformatics software was used to predict the possible target genes of differentially expressed miRNA in the TGF-?/BMP signaling pathway,and the expression of target genes in MC3T3-E1 cells under stretch stress was verified by western blot.Target gene validation was performed by miRNAs transfection,qRT-PCR,Western Blot,and dual luciferase reporter assays.The change of cell proliferation ability was detected by CCK8 method in miRNA transfected cells.The expression of osteogenic differentiation marker factors was detected by qRT-PCR,and the role of miRNA in MC3T3-E1 cells proliferation and osteogenic differentiation was explored.Results:1.The results of miRNAs chip showed that there were 44 miRNAs differentially expressed in the stress-loading group compared with the control group(p < 0.05).The results of qRT-PCR showed that the expression of miR-132-3p was most significant and its expression was up-regulated in the stress-loading cells.2.Bioinformatics software analysis showed that the possible target genes of miR-132-3p in TGF-?/BMP signaling pathway were Smad2 and Smad5,and the results of Western Blot also showed the expressions of Smad2,Smad5,p-Smad2 and p-Smad5 were reduced after stress loading.The results of qRT-PCR and western blot showed that overexpression of miR-132-3p could inhibit the expression of Smad5 protein,but the effect on Smad5 m RNA was not obvious.Dual luciferase experiments showed that Smad5 is an effective target gene for miR-132-3p.Functional studies showed that when miR-132-3p overexpressed in MC3T3-E1 cells,the expression levels of cell proliferation and osteogenic differentiation factors,such as ALP,OCN and Runx2 decreased significantly.Conclusion:1.12% tensile stress can activate differential expression of multiple miRNAs in MC3T3-E1 cells,miR-132-3p expression is most significant;2.Mi R-132-3p may inhibit the proliferation and osteogenic differentiation of MC3T3-E1 cells by targeting Smad5.