Experimental Study On The Effect Of Hsa-miRNA-223-3p On Osteogenic Differentiation Cells Of Human Bone Marrow Mesenchymal Stem Cells

Objective: To investigate the regulatory effect of hsa-miR-223-3p on BMSCs osteogenic differentiation cells by the isolation,purification and identification of human bone marrow mesenchymal stem cells(BMSCs),as well as the induction of osteogenic differentiation of BMSCs in vitro,overexpression of hsa-miR-223-3p.Methods: 10 mL of fresh human bone marrow was extracted,and anticoagulated by heparin(2500 U/mL,0.5 mL),and then injected into a test tube containing a Percoll solution,and mononuclear cells were separated and purified by Percoll density gradient centrifugation.BMSCs were cultured in 10% FBS DMEM complete medium.The expression of related surface antigen markers on adherent human bone marrow mesenchymal stem cells was identified by immunocytochemistry.Differentiation of human BMSCs into osteoblasts was induced by dexamethasone,sodium ?-glycerophosphate,and vitamin C.On the 7th,14 th,and 21 st day of cell-induced culture,ALP levels were detected and alizarin red staining was performed.In osteoblasts,overexpressing hsa-miR-223-3p,RT-qPCR was used to detect the mRNA level of Wnt5 a,OPG,RUNX2.The expression of Wnt5 a,OPG and RUNX2 was detected by Western blotting.The binding sites of hsa-miR-223-3p on Wnt5 a were found by bioinformatics software.PmirGLO/Wnt5a-3'UTR and pmirGLO/Wnt5a-3'UTR mut plasmids were constructed and co-transfected with hsa-miR-223-3p mimcs/NC to 293 T cells to verify the targeting relationship between hsa-miR-223-3p and Wnt5 a by dual luciferase reporter gene system.Results: 1.The cells isolated by Percoll density gradient centrifugation have a flat spindle shape,similar to fibroblasts;the nucleus is relatively large,one or more nucleoli,and the cytoplasm is abundant.The results of immunocytochemistry showed that CD 34,CD 45 was negative and CD 44 was positive,suggesting that the cultured cells were human bone marrow mesenchymal stem cells,which guaranteed the accuracy of the subjects.2.With the prolongation of osteogenic induction time,the level of ALP and the deposition of dark red or reddish-brown calcium salt nodules gradually increased.3.In osteoblasts,hsa-miR-223-3p decreased the mRNA and protein levels of Wnt5 a,OPG,and RUNX2(P<0.05).4.hsa-miR-223-3p directly targets the 3'UTR region of Wnt5 a,reducing the activity of luciferase of Wnt5 a.This target effect disappeared when the seed sequence of hsa-miR-223-3p binding to Wnt5 a was mutated.When hsa-miR-223-3p was blocked,the activity of luciferase of wild-type Wnt5 a increased,while the activity of luciferase of mutant Wnt5 a did not change.Conclusion: The cells isolated and cultured by Percoll density gradient centrifugation have the characteristics of human bone marrow mesenchymal stem cells.hsa-miR-223-3p can affect the Wnt signaling pathway in BMSCs osteoblast differentiation cells.hsa-miR-223-3p binds directly to Wnt5 a,and Wnt5 a acts as a downstream target gene of hsa-miR-223-3p,which is negatively regulated by it.