GATA4 Targets MiR-144-3p To Inhibit Osteogenic Differentiation

Objective: To investigate the role of miR-144-3p in osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)and the regulatory mechanism of GATA4 on miR-144-3p.Methods: 1?Induced rat osteogenetic differentiation of mesenchymal stem cells,after induction of 0,7,14,21 days respectively for ALP(alkaline phosphatase staining)and ARS(alizarin red staining),at the same time using the real-time fluorescence Quantitative polymerase chain reaction(policy Real-time PCR,hereinafter referred to as the q RT-PCR)determination of miR-144-3p,osteogenesis sign gene ALP and,Runx2(Runt related transcription factor 2)m RNA expression;2.BMSCs cells were transfected with a miR-144-3p mimic,inhibitor and their negative control,and the expression levels of miR-144-3p m RNA were determined by q RT-PCR at 48 h after transfection.The transfected cells were further induced by osteogenesis for 48-72 hours.3.Adenovirus carrying adgata-4 was used to infect BMSCs,and the miRNA contents of GATA-4 and miR-144-3p were determined by q RT-PCR after 48 h of infection,and the relationship between GATA-4 expression and miR-144-3p m RNA expression was analyzed.Through the analysis of biological information,view GATA-4 may be targeted on the miR-144-3 p precursor-568 and-969 area,build plasmid containing a role site-568 P1,contains two role site-568 and 969-the plasmid of P2,p GL3 basic plasmid as control group,respectively transfection tools 293 t cells,transfection 48 h after hours,using dual luciferase activity test to verify the GATA-4for the role of miR-144-3 p site.Results:1.In the process of osteogenic induction and differentiation of bone marrow mesenchymal stem cells,the deeper the alkaline phosphatase staining and alizarin red staining were with the extension of induction time,the m RNA expressions of osteogenic marker genes ALP and Runx2 also increased,while the m RNA expressions of miR-144-3p decreased.2.The m RNA and protein expressions of the osteogenic marker genes ALP,Runx2 and Runx2 in the miR-144-3p group after transfection were significantly decreased compared with the inhibited group.3.The m RNA levels of GATA-4 and miR-144-3p in the Ad GATA-4 group were significantly higher than those in the negative control group and the blank control group.The immunofluorescence activity of plasmid P1 and P2 group was significantly increased compared with that of transfected basic plasmid group.Conclusion: 1.Mi R-144-3p plays a negative regulatory role in the differentiation of osteoblasts.2.The GATA4 regulatory sites were located in the 568 and 969 regions of the miR-144 promoter.3.GATA4 inhibits BMSCs osteogenic differentiation by targeting the expression of miR-144-3p.