miR-363在脆性骨折患者血中的表达及对MC3T3-E1细胞成骨分化及增殖的影响

Expression of miR-363 in blood of patients with brittle fracture and its effect on osteogenic differentiation and proliferation of MC3T3-E1 cells

目的研究miR-363在脆性骨折患者血中的表达及对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)成骨分化与增殖的影响。方法收集脆性骨折患者外周血,采用荧光定量PCR检测患者miR-363、矮小相关转录因子2(RUNX2)、骨钙素(OCN)、细胞周期蛋白D1(CCND1)、连环蛋白β1(CTNNB1)和骨髓细胞瘤癌基因(MYC)表达。采用茜素红S染色与噻唑蓝检测过表达miR-363对细胞成骨分化及增殖的影响。采用双荧光素酶报告基因与Western blotting检测miR-363对Dickkopf相关蛋白1(DKK1)的调控作用。结果miR-363在脆性骨折患者外周血中的表达水平低于正常健康者,而术后1周、2周及4周外周血中的表达水平高于术前并呈上调趋势(P<0.05);模拟物组MC3T3-E1细胞中miR-363表达水平高于阴性对照组(P<0.05);模拟物组MC3T3-E1细胞矿化程度、RUNX2及OCN mRNA表达水平及1天、2天、3天光密度(OD)值高于阴性对照组(P<0.05);DKK1是miR-363的靶基因,模拟物组MC3T3-E1细胞中DKK1蛋白表达水平低于阴性对照组,而CCND1、CTNNB1及MYC mRNA表达水平高于阴性对照组(P<0.05)。结论miR-363可能通过调控靶基因DKK1与Wnt/β-catenin信号通路促进MC3T3-E1细胞成骨分化与增殖。

Aim To investigate the expression of miR-363 in the blood of patients with brittle fracture and the effect of miR-363 on the osteogenic differentiation and proliferation of mouse embryonic osteoblast precursor cells(MC3 T3-E1).Methods The peripheral blood of patients with fragility fractures were collected,and the expression of miR-363,runt-related transcription factor 2(RUNX2),osteocalcin(OCN),cyclin D1(CCND1),catenin beta 1(CTNNB1)and myelocytomatosis oncogene(MYC)were detected by fluorescence quantitative PCR.The effect of overexpression of miR-363 on osteogenic differentiation and proliferation of cells was detected by alizarin red S staining and thiazole blue.The regulation of mi R-363 on Dickkopf-1(DKK1)was detected by dual luciferase reporter gene and Western blotting.Results The expression level of mi R-363 in peripheral blood of fragility fracture patients was lower than that of normal healthy individuals,but its expression level in peripheral blood of these patients at 1 week,2 weeks,and 4 weeks after operation was higher than that of pre-operation and showed an upward-regulated trend(P<0.05);the expression level of mi R-363 in MC3 T3-E1 cells in the mimic group was higher than that in the negative control group(P<0.05);the degree of mineralization,the m RNA expression levels of RUNX2 and OCN,and optical density(OD)at 1 day,2 days,and 3 days in MC3 T3-E1 cells in the mimic group were higher than those in the negative control group(P<0.05);DKK1 is the target gene of mi R-363,the expression level of DKK1 protein in MC3 T3-E1 cells in the mimic group was lower than that in the negative control group,while the m RNA expression levels of CCND1,CTNNB1 and MYC were higher than that in the negative control group(P<0.05).Conclusion mi R-363 may promote osteogenic differentiation and proliferation of MC3 T3-E1 cells by regulating target gene DKK1 and Wnt/β-catenin signaling pathway.