MiR-21 Induced Canine BMSCs Osteogenic Differentiation In Vitro

Objective To explore the effect of miR-21 on the osteogenic differentiation of Labrador dog bone marrow mesenchymal stem cells(BMSCs)in vitro.Methods Labrador dog BMSCs were cultured in vitro using whole bone marrow adherent method,Lenti-miR-21-Luciferase and Lenti-Lac Z-Luciferase lentiviral vector were constructed and then were transfected into canine BMSCs.The study was divided into miR-21 experimental group and Lac Z control group.After 4 days of transfection,observe the cells under inverted microscope.Detect the transfection efficiency of lentivirus by observing fluorescence intensity of tracer reporter Luciferase through bioluminescence imaging methods.MTT colorimetric assay was used to detect the proliferation of BMSCs after lentivirus transfection.The cell scratch test was used to compare the migration ability of BMSCs after lentiviral vector transfection.The m RNA and protein of BMSCs were extracted on 0,1,4,7,and 14 days.The expression of osteocalcin(OCN)and osteopontin(OPN),the key factors of osteogenic differentiation of BMSCs,were detected by Real time-PCR and Western blot.Results Microscopic observation shows the shape of cell is uniform,mostly spindle-shaped or long spindle and the cells were arranged in a spiral or radial fusion,also grow well.The transfection efficiency of BMSCs was the highest and the result of Luciferase's fluorescence luminescence intensity test suggest that the tracer gene Luciferase is successfully linked in BMSCs when the MOI value is 10.The mean reduction of scribe width in miR-21 group was 372.33 ?m while that of Lac Z control group was only 161.33 ?m,P<0.001(F = 0.10),differences were statistically significant.The result suggests the crawling ability of BMSCs cell in miR-21 experimental group increased significantly.Alizarin Red S Calcium nodule staining and ALP staining results suggest the expression of calcium nodules and ALP in BMSCs was significantly increased in miR-21 group comparing with Lac Z group.Real time-PCR quantitative results show BMSCs in the miR-21 experimental group were significantly expressed in OCN and OPN genes at 7 days after lentivirus transfection.At the 14 th day after transfection,the two genes were still highly expressed.The results showed that miR-21 could significantly enhanced the expression of osteogenic factors in BMSCs(P<0.05).Western blot analysis shows that miR-21 can significantly increase the expression of OCN and OPN protein in osteogenic differentiation of BMSCs(P<0.05),and the expression trend is close to that of m RNA.Conclusion miR-21 played as a carrier with lentivirus,can successfully transfected BMSCs and efficiently enhance the expression of osteogenesis-related factors in cells.The miR-21 could significantly promote the osteogenic differentiation of BMSCs,laying the foundation for further in vivo experiments.