| BACKGROUNDThe aging of our country has become increasingly serious,bone diseases as a high incidence of diseases of the elderly population has attracted the attention of the medical community.With the continuous study of osteoblastic differentiation mechanism of osteoblasts,it will help us to provide new ideas for the treatment of bone diseases.More and more studies have shown that miRNAs play an important regulatory role in bone metabolism balance,directly participating in the occurrence process of many bone diseases,however,the role and mechanism of miR-223-5p in osteogenic differentiation has not been reported.OBJECTIVEmiR-223-5p was selected as the object of study to explore the effect of miR-223-5p on osteoblastic differentiation of MC3T3-E1 cells,and further explored its mechanism of action,providing new ideas and support for the study of related mechanisms of osteoblastic differentiation and the treatment of bone diseases.METHODS1.Effect of miR-223-5p on osteoblastic differentiation of MC3T3-E1 cells(1)MC3T3-E1 cells were cultured in complete medium and osteogenic differentiation induction medium respectively,and the expression of miR-223-5p,ALP,OCN and Runx2 before and after induction were detected by real-time fluorescence quantitative PCR.The expression of ALP,OCN and Runx2 were detected by Western Blot,and the activity of ALP was detected.(2)In order to detect the effect of miR-223-5p on osteogenic differentiation of MC3T3-E1 cells,the mimics or inhibitor of miR-223-5p was transfected into MC3T3-E1 cells to construct a miR-223-5p overexpression or low expression model,and the overexpression and interference efficiency were detected by real-time fluorescence quantitative PCR.After transfection,the expressions of ALP,OCN and Runx2 were detected by real-time fluorescence quantitative PCR,and the formation of mineralized nodules was observed by ALP activity detection and alizarin red staining.2.Mechanism of miR-223-5p regulating HDAC2 expression and promoting osteoblastic differentiation of MC3T3-E1 cells(1)Combined with bioinformatics and online database analysis,the downstream target genes of miR-223-5p were predicted.(2)Transfected with miR-223-5p mimics or inhibitor in MC3T3-E1 cells,the miR-223-5p overexpression or low expression model was established,and the expression level of HDAC2 was detected by real-time fluorescence quantitative PCR and Western Blot.(3)The expression of HDAC2 was down-regulated by Si-HDAC2 transfection in MC3T3-E1 cells,and the expression of HDAC2 was detected by real-time fluorescence quantitative PCR and Western Blot.After successful transfection,the expressions of ALP,OCN and Runx2 were detected by real-time fluorescence quantitative PCR and Western Blot,and the formation of mineralized nodules was observed by ALP activity detection and alizarin red staining.(4)MC3T3-E1 cells were treated with CAY 10683,a HDAC inhibitor,at different concentrations,and the expressions of HDAC2,ALP,OCN and Runx2 were detected by real-time fluorescence quantitative PCR and Western Blot.(5)mir-223-mimics and HDAC2 overexpressed plasmids were co-transfected in MC3T3-E1 cells,and the expressions of ALP,OCN and Runx2 were detected by real-time fluorescence quantitative PCR and Western Blot.The formation of mineralized nodules was observed by ALP activity detection and alizarin red staining.3.In vivo study on the regulation of miR-223-5p in osteoblastic differentiation of MC3T3-E1 cellsMC3T3-E1 cells were transfected with S1-HDAC2 to form a mixture with HA-TCP and transplanted into nude mice surgically.HE staining and Masson staining were performed on tissue sections to study the effect of miR-223-5p on osteoblastic differentiation of MC3T3-E1 cells in vivo from the perspective of histology.RESULTS1.Effect of miR-223-5p on osteoblastic differentiation of MC3T3-E1 cells(1)Osteoblastic differentiation induction medium was able to induce osteoblastic differentiation of MC3T3-E1 cells.Compared with complete medium,the expressions of miR-223-5p,ALP,OCN and Runx2 genes were increased,the expressions of ALP,OCN and Runx2 proteins were increased,and the ALP activity was increased.(2)After transfection with miR-223-5p mimics,gene expressions of miR-223-5p,ALP,OCN and Runx2 were increased,ALP activity was increased,and calcium nodule formation was increased in MC3T3-E1 cells.After transfection with miR-223-5p inhibitor,the expression of miR-223-5p,ALP,OCN and Runx2 genes decreased,ALP activity decreased,and calcium nodule formation decreased in MC3T3-E1 cells.2.Mechanism of miR-223-5p regulating HDAC2 expression and promoting osteoblastic differentiation of MC3T3-E1 cells(1)Combined with bioinformatics and online database analysis,the downstream target gene of miR-223-5p was HDAC2.(2)After transfection with miR-223-5p mimics,the gene and protein expression of HDAC2 decreased,while after transfection with miR-223-5p inhibitor,the gene and protein expression of HDAC2 increased.(3)After transfection of si-HDAC2,the expression of HDAC2 gene and protein decreased.When HDAC2 expression was down-regulated,ALP,OCN,and Runx2 gene and protein expressions increased,ALP activity increased,and calcium nodule formation increased in MC3T3-E1 cells.(4)CAY 10683 inhibited the expression of HDAC2,which presented a dose-dependent relationship with CAY 10683.With the increase of CAY 10683 dose,the expression of HDAC2 decreased and the expression of ALP,OCN and Runx2 increased.(5)When MC3T3-E1 cells were co-transfected with mir-223-mimics and HDAC2 overexpressed plasmids,compared with mimics-nc and HDAC2 overexpressed plasmids,gene and protein expressions of ALP,OCN and Runx2 increased,ALP activity increased,and calcium nodule formation increased in MC3T3-E1 cells.3.In vivo study on the regulation of miR-223-5p in osteoblastic differentiation of MC3T3-E1 cellsThe results of HE staining and Masson staining of tissue sections showed that the osteoblast differentiation ability of MC3T3-E1 cells transfected with si-HDAC2 was stronger than that of si-nc group in vivo.CONCLUSIONOsteoblastic differentiation induction medium promoted the osteoblastic differentiation of MC3T3-E1 cells,and miR-223-5p expression was increased during the osteoblastic differentiation of MC3T3-E1 cells.miR-223-5p can promote the osteogenic differentiation of MC3T3-E1 cells.This study confirmed that miR-223-5p promoted the osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of its target gene HDAC2.Down-regulation of HDAC2 expression can improve the osteogenic differentiation ability of MC3T3-E1 cells in vivo. |
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