MiRNA Profiling Analysis During Osteogenic Differentiation Of MC3T3-E1 Cells Subjected To Compressive Stress And Functional Verification Of MiR-222-3p

Research background and objectiveThe idea that stretch force promotes osteogenesis and compressive stress induces bone absorption has been widely accepted,but in recent years studies have reported the compressive stress can also promote osteogenesis,preliminary research results displayed the 2 g/cm2 was the best force value for osteoblasts to promote osteogenesis,this suggests that compressive stress may be a key factor affecting the function of osteoblasts.In other words,under the effect of light compressive stress,the function of osteoblasts is best,and the function of osteoblasts is regulated by a variety of transcription factors,among which miRNA is an important gene post-transcriptional regulator.So this research adopts the 2 g/cm2 as the experimental group.By constructing the model of MC3T3-E1 cells in vitro 3D culture and stress loading device,the expression profile of microRNA in osteoblasts were explored,and the possible target genes of miR-222-3p and its regulatory mechanism on osteogenic differentiation were further explored.Methods1.Establish three-dimension(3D)culture mode of MC3T3-E1 cells and the application of compressive loading in vitro,Calcein AM/PI and CCK8 experiment were applied to test the feasibility of this model.2.0g/cm2 group as control group,2g/cm2 group as experimental group.After loading 24 h,the effcts of stress loading on the expression of osteogenic differentiation related genes were investigated via qRT-PCR and ALP staining;differentially expressed miRNAs between two groups were detected by RNA-seq and verified by qRT-PCR.3.The identification of target miRNA was based on the results of qRT-PCR.The target genes of miRNA were predicted by using bioinformatics software.The effects of miR-222-3p on osteogenic differentiation of MC3T3-E1 cells were determined by the transient transfection that induced the target miRNA overexpression and inhibition,verification of the target gene and functional verification of miR-222-3p were applyed with the qRT-PCR,Western blot and double-luciferase reporter assay.Results1.The calcein AM/PI double staining of living and dead cells showed that the cells had a good survival rate in rat tail collagen;CCK8 experiment showed that this model didn't influence the activity of osteoblasts.2.Compared with the control group,qRT-PCR and ALP staining experiments showed that compressive stress improved the osteogenic differentiation of MC3T3-E1;the results of miRNA expression profile showed that 74 miRNAs were expressed differently,43 up-regulated and 31 down-regulated;qRT-PCR experiments showed that miR-222-3p was down-regulated and the change of it was the most obvious,the trend was consistent with the profile results,then the target genes were predicted might be Runx2 and BTG2.3.Western blot experiments showed that the change of Runx2 expression was contrary to the change of miR-222-3p,but the BTG2 expression was declined.Therefore,the possibility of BTG2 as the target gene was excluded.miR-222-3p was overexpressed in MC3T3-E1 cells,Western blot showed that the expression of Runx2 declined,The double-luciferase experiment also confirmed that miR-222-3p and Runx2 can be effectively combined.The functional study showed that the expression level of osteogenic related genes declined significantly when miR-222-3p was overexpressed.Conclusions1.The expression of multiple miRNAs changed and miR-222-3p expression decreased mostly in the process of osteogenic differentiation of MC3T3-E1 cells mediated by compressive stress.2.miR-222-3p may inhibit the osteogenic differentiation through the target gene Runx2.