Atf7ip is a negative regulator of bone morphogenetic protein 2 promoting osteogenic differentiation in mouse embryonic adult cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Whether activating transcription factor 7 interacting protein(Atf7ip)is involved in the regulation in osteogenic differentiation is still controversial,and studying its impact on osteogenic differentiation and its specific mechanisms is of great significance. OBJECTIVE:To investigate the effect of Atf7ip on bone morphogenetic protein 2 promoting osteogenic differentiation of mouse embryonic osteoblast precursor cells(MC3T3-E1). METHODS:MC3T3-E1 cells cultured in vitro were divided into three groups:normal group,interference group(NC-siRNA group,Atf7ip-siRNA group),and high expression group(CMV-VC group and CMV-Atf7ip group),and were transfected for 24 hours,and then treated with 200 ng/mL bone morphogenetic protein 2 for 0,12,24,and 48 hours,respectively.qRT-PCR was used to detect the mRNA expression levels of Atf7ip,alkaline phosphatase,osteocalcin,type I collagen α1 in the cells of each group.Western blot assay was used to detect the protein expression of osteogenic differentiation markers Sp7 and Runx2,and the expression of Atf7ip binding molecule SETDB1,histone H3 and H3K9me3.Alkaline phosphatase activity was detected by alkaline phosphatase staining. RESULTS AND CONCLUSION:(1)With the increase of bone morphogenetic protein 2 treatment time,the protein and mRNA expression of Atf7ip decreased,while the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and alkaline phosphatase increased significantly(P<0.05).There was no significant change in the protein expression of Atf7ip binding molecule SETDB1.(2)Compared with the NC-siRNA group,the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and type I collagen α1 were significantly up-regulated(P<0.05),and alkaline phosphatase activity was significantly enhanced;and H3K9 methylation significantly decreased in the Atf7ip-siRNA group(P<0.05).(3)Compared with the CMV-VC group,the protein expression of Sp7 and Runx,as well as mRNA expression of osteocalcin,alkaline phosphatase,and type I collagen α1 was significantly downregulated(P<0.05),and the alkaline phosphatase activity was significantly reduced in the CMV-Atf7ip group,while the H3K9 methylation protein in the CMV-Atf7ip group was significantly upregulated compared to the control group(P<0.05).(4)In conclusion,Atf7ip expression was decreased during bone morphogenetic protein 2-induced osteogenic differentiation of MC3T3-E1,and osteogenic differentiation was significantly increased after knockdown of Atf7ip.Overexpression of Atf7ip significantly weakened osteogenic differentiation,indicating that Atf7ip is a negative regulatory factor of bone morphogenetic protein 2 promoting osteogenic differentiation of MC3T3-E1 cells.