MiR-21-5p Promotes Osteoblast Differentiation Via Downregulating SMAD7,in Response To Hypoxia

ObjectiveBy miR-21-5p transfection into osteoblast MC3T3-E1,to test at different time after transfection of osteoblasts transfection concentration and transfection of microRNAs-21-the change of 5 p content,while detecting osteoblast differentiation after transfection protein: osteocalcin,P1 NP and RUNX2 content changes;At the same time at different times and transfection concentration detection transfection osteoblast MC3T3-E1 osteoblast after matrix mineralization degree,thus reveals the miR-21-5p and the correlation of osteoblast differentiation and mineralization,for clinical treatment of fracture healing,osteoporosis drug research and development to provide new targets,has an important value.MethodsThe MC3T3-E1 cells were obtained from ATCC,cultured for 24 h,then transfected with miR-21-5p mimics or recombinant plasmid by Lipofectamine 2000 Transfection Reagent.The relative mRNA level of the osteoblast differentiation-associated markers was measured by RT-PCR.U6 was used as internal control.The luciferase gene and the 3’ UTR of SMAD7 were amplified by RT-PCR with Q5 High-Fidelity DNA Polymerase after construction of the recombinant plasmid and the luciferase reporter assay.The gene of 3’ UTR of SMAD7 or mutant 3’ UTR of SMAD7 were cloned into the pLcu vector just at the downstream of the luciferase reporter.After co-transfecting,collected the cells and examined the relative luciferase activity assayed with the Dual-Luciferase Assay kit.ECM mineralization assay and Alkaline Phosphatase Activity were used to quantify the influence of miR-21-5p.Western Blot was used to measure the expression of SMAD7 on protein level.ResultsRT-PCR showed that osteoblast differentiation-associated markers as well as the level of miR-21-5p were up-regulated in mouse osteoblast-like MC3T3-E1 cells post transfection of miR-21-5p(p<0.01).The level of miR-21-5p was significantly increased at 3h post transfection,stable from 3h to 24 h,compared to the control group(p<0.01).The mRNA level of differentiation-associated markers such like osteocalcin,P1 NP and RUNX2,with β-actin as internal control,were significantly increased in the MC3T3-E1 cells post transfection with miR-21-5p mimics for 12 hours,compared to the scramble groups(p<0.01).miR-21-5p upregulates the extracellular matrix(ECM)mineralization in MC3T3-E1 cells.Besides the image,we also quantified the OD450 value of the cells.The OD450 value was increased in the cells transfected with 30 nM(p<0.05)or 60 nM(p<0.01)miR-21-5p mimics,as compared with the scramble group.Alkaline Phosphatase(ALP)is a marker in the early stage of osteogenesis,the relative ALP activity in the miR-21-5p mimics-transfected MC3T3-E1 cells was much higher than the scramble group,especially at the concentration of 60 n M miR-21-5p mimics(p<0.05,p<0.01).miR-21-5p targets the 3’ UTR of SMAD7 in the MC3T3-E1 cells,as the experiment aligning the sequence of miR-21-5p and 3’ UTR of SMAD7 mRNA,and found that 8 bases were exactly complementary.The relative luciferase activity was significantly decreased in the cells post transfecting with pLuc-SMAD7 3’ UTR and miR-21-5p mimics(30nM or 60nM),compared to the cells transfected with pLuc-SMAD7 3’ UTR and scramble(p<0.05).However,the relative luciferase activity in the pLuc-SMAD7 3’ UTR mut-transfected MC3T3-E1 cells showed no significant difference from the cells co-transfected with 30 nM or 60 nM miR-21-5p mimics and scramble(ns: no significance).The results demonstrated that the miR-21-5p mimics reduced the relative luciferase activity by targeted the 3’ UTR of SMAD7.The expression level of SMAD7 was down-regulated by miR-21-5p.The relative luciferase activity was reduced by the targeting of the miR-21-5p and 3’ UTR SMAD7 in the luciferase-miR-21-5p-mimics co-transfection system,therefore,miR-21-5p may play a role in modulating the expression level of SMAD7 in the MC3T3-E1 cells.The expression levels of SMAD7 protein in the cells transfected with 30 nM or 60 nM miR-21-5p mimics were significant decreased compared with scramble groups.Furthermore,the higher concentration of miR-21-5p mimics led to the lower expression of SMAD7(p<0.01)Conclusion1.miR-21-5p promotes the generation of osteoblast differentiation markers(osteocalcin and P1 NP,RUNX2,alkaline phosphatase).2.miR-21-5p plays a role in promoting osteoblast differentiation through the 3’ UTR end of SMAD7.3.miR-21-5p may be an important molecular target for promoting osteoblast differentiation,inhibiting osteoporosis and promoting fracture healing.