miR-25/TWIST1调控通路在成骨细胞矿化中的作用研究

Study on the role of miR-25/TWIST1 regulatory pathway in osteoblast mineralization

目的探究miR-25在成骨细胞分化过程中的作用及其作用机制。方法β-甘油磷酸(β-glycerophosphate,β-GP)和抗坏血酸(aseorbic acid)诱导成骨细胞矿化,茜素红(Alizarin Red S)染色实验观察成骨细胞的矿化情况。生物信息学运算预测miR-25的靶基因。实时定量PCR法检测miR-25以及其他相关基因的表达。过表达实验和抑制实验用于研究miR-25在成骨细胞分化中的作用及其与靶基因之间的相互关系。荧光素酶报告基因实验验证miR-25与其靶基因之间的结合。结果 miR-25的表达随成骨分化过程而逐渐升高。过表达miR125通过转录后调控其靶基因TWIST1的表达来促进成骨分化。作为调控成骨细胞分化的的转录因子,扭曲基础螺旋—环—螺旋蛋白1(TWIST1)是miR-25的靶基因。结论 miR-125通过作用于靶基因TWIST1在成骨细胞的分化中发挥了重要的调控作用,表明调控miR-25的表达,可能成为骨代谢疾病新的治疗靶点。

Objective To explore the role of miR-25 in the process of osteoblast differentiation and its mechanism.Methods β-glycerophosphate（β glycerophosphate,β-GP） and ascorbic acid（ascorbic acid） induced osteoblast mineralization,Alizarin Red（Alizarin Red S） experiment were performed to observe stained cells into bone mineralization.Bioinformatics computing predicted target genes of miR-25.Real-time quantitative PCR was used to detect the expression of miR-25 and other related genes.Overexpression experiments and inhibitions experiments were used to investigate the relationship between of miR-25 in osteoblast differentiation and its relationship with target gene.Luciferase gene reporter experiments were used to demonstrate miR-25 binding to its target genes.Results The expression of miR-25 increased gradually in the process of osteogenic differentiation.Overexpression of miR-25 promotes osteogenic differentiation through post transcriptional regulate TWIST1 expression of its target genes.As a transcription factor that regulates the differentiation of bone cells,TWIST1 is a target gene of miR-25.Conclusion miR-25 plays an important role in the differentiation of osteoblasts by acting on target gene TWIST1,which indicates that the expression of miR-25 may be a new therapeutic target for bone metabolism.