The Mechanism Of DMP1 And BMP1 Co-Overexpression System Regulating Osteoblast Differentiation

Objective: Dentin matrix protein 1(DMP1)and bone morphoprotein 1(BMP1)have been shown to play important roles in osteoblast differentiation and bone repair and regeneration,respectively.However,the interaction between DMP1 and BMP1 and its specific regulatory mechanism on osteoblast differentiation are still unclear.The purpose of this study was to explore the effect of co-overexpression of DMP1 and BMP1 on the osteogenic differentiation ability of MC3T3-E1 cells,and preliminarily explore its potential regulatory mechanism,so as to provide a new strategy for clinical treatment of maxillofacial bone defects.Materials and methods: 1.Detection of osteogenic differentiation ability of MC3T3-E1cells: MC3T3-E1 cells were induced to differentiate in osteoblastic culture medium,the ability of osteogenic differentiation was detected by alkaline phosphatase(ALP)and alizarin red S(ARS)staining,and the expression of Runt related transcription factor 2(RUNX2)was detected by qRT-PCR;2.To detect the effect of co-overexpression of DMP1 and BMP1 on osteogenic differentiation of MC3T3-E1 cells: the following plasmids were constructed:(1)Empty control enhanced green fluorescent protein particle(pEGFP)(2)DMP1 fusion expression EGFP plasmid(p DMP1/EGFP)(3)BMP1 fusion expression EGFP plasmid(pBMP1/EGFP)(4)BMP1-DMP1 co-expression fusion EGFP plasmid(pBMP1+DMP1/EGFP),and transfected into MC3T3-E1 cells.Western Blot and qRT-PCR detected the mRNA and protein expression of osteogenic markers in MC3T3-E1 cells.ALP staining detected the positive expression of ALP,and ARS staining detected the formation of mineralized nodule;3.Bioinformatics analysis and prediction of target genes: use STRING database to predict the corresponding target genes of DMP1 and BMP1,and verify it;4.To detect the effect of glucose-regulated protein 78(GRP78)on the osteogenic differentiation of MC3T3-E1 cells: construct GRP78 siRNA(si-GRP78)and its negative control siRNA,construct GRP78 over-expression plasmid and its empty control plasmid,and transfect MC3T3-E1 cells.Western blot and qRT-PCR detected the mRNA and protein expression of osteogenic markers in MC3T3-E1 cells.ALP positive expression was detected by ALP staining,and the formation of mineralized nodules was detected by ARS staining;5.To detect the regulatory effect of DMP1 and BMP1 cooverexpression on GRP78: set up the following experimental groups:(1)empty control group(2)pBMP1+DMP1/EGFP transfection group(3)si-GRP78 transfection group(4)pBMP1+DMP1/EGFP and si-GRP78 co-transfection group,and transfected into MC3T3-E1 cells.Western blot detected the protein expression of osteogenic markers in MC3T3-E1 cells,ALP staining detected the positive expression of ALP,ARS staining detected the formation of mineralized nodules,and CCK-8 detected the proliferative activity of cells.Results: 1.Detection of osteogenic differentiation ability of MC3T3-E1 cells: ALP and ARS staining showed that the osteogenic differentiation of MC3T3-E1 cells increased after osteoinduction.QRT-PCR showed that the expression of RUNX2 was up-regulated after osteoinduction of MC3T3-E1 cells,indicating that MC3T3-E1 cells had good osteogenic differentiation ability;2.To detect the effect of co-overexpression of DMP1 and BMP1 on osteogenic differentiation of MC3T3-E1 cells: western blot and qRT-PCR results showed that the co-overexpression of DMP1 and BMP1 significantly promoted the expression of osteogenic markers mRNA and protein in MC3T3-E1 cells.ALP and ARS staining showed that the co-overexpression of DMP1 and BMP1 significantly promoted the expression of ALP and the formation of mineralized nodules in MC3T3-E1cells;3.Bioinformatics analysis and prediction of target genes: STRING database found that GRP78 may be the significant target gene of DMP1 and BMP1,and the results of qRT-PCR and western blot showed that the co-overexpression of DMP1 and BMP1 significantly promoted the protein and mRNA expression of GRP78 in MC3T3-E1 cells;4.To detect the effect of GRP78 on the osteogenic differentiation of MC3T3-E1 cells:qRT-PCR and western blot showed that GRP78 knockdown significantly inhibited the mRNA and protein expression of osteoblast-related markers in MC3T3-E1 cells.ALP and ARS staining showed that GRP78 knockdown significantly inhibited the ALP positive expression and the formation of mineralized nodules,while GRP78 overexpression showed the opposite results;5.To detect the regulatory effect of DMP1 and BMP1 coexpression on GRP78: the results of western blot,ALP and ARS staining showed that the co-overexpression of DMP1 and BMP1 rescued the osteogenic inhibition of GRP78 knock down on MC3T3-E1 cells.The results of CCK-8 experiment showed that the cooverexpression of DMP1 and BMP1 promoted the proliferation of MC3T3-E1 cells,and GRP78 knockdown inhibited the proliferation of MC3T3-E1 cells,while the cooverexpression of DMP1 and BMP1 could reverse the inhibition of GRP78 knockdown on the proliferation of MC3T3-E1 cells.Conclusion: This study showed that the co-overexpression of DMP1 and BMP1 had the most significant promoting effect on the osteogenic differentiation of MC3T3-E1 cells.At the same time,GRP78 can also positively regulate the osteogenic differentiation of MC3T3-E1 cells,and can be a potential target of DMP1 and BMP1.