微RNA-218通过抑制RUNX2及Ⅰ型胶原基因表达延缓后纵韧带骨化

MicroRNA-218 delay ossification of posterior longitudinal ligament cells by inhibiting expression of RUNX2 and typeⅠ collagen

目的探讨微RNA-218(mi RNA-218)对后纵韧带骨化症(OPLL)患者原代后纵韧带细胞骨化的影响及其作用机制。方法原代培养5例OPLL患者及5例非OPLL者的韧带细胞,比较2组细胞miRNA-218的表达差异。利用agomir过表达或antagomir抑制OPLL患者韧带细胞中miRNA-218的表达水平后进行成骨诱导,通过检测茜素红染色水平、碱性磷酸酶活性及成骨相关基因的表达验证miRNA-218对韧带细胞骨化的作用。采用Target scan预测miRNA-218的靶基因,并采用双荧光素酶报告基因实验验证miRNA-218的靶向作用。结果 OPLL患者韧带细胞miRNA-218的表达水平低于非OPLL者,差异有统计学意义(P <0.05)。过表达miRNA-218后OPLL患者韧带细胞茜素红染色水平、碱性磷酸酶活性及成骨相关基因的表达均低于对照组;抑制miRNA-218后OPLL患者韧带细胞茜素红染色水平、碱性磷酸酶活性及成骨相关基因的表达均高于对照组;差异均有统计学意义(P <0.05)。Target scan预测miRNA-218靶基因可能为RUNX2和Ⅰ型胶原(COL1A1),双荧光素酶报告基因实验显示miRNA-218能降低RUNX2及COL1A1基因的荧光素酶活性水平。结论 miRNA-218能明显抑制原代后纵韧带细胞的骨化反应,其作用机制与抑制RUNX2和COL1A1基因表达相关。

Objective To investigate the function and mechanism of microRNA-218(miRNA-218) in regulating the ossification of posterior longitudinal ligament(OPLL) cells. Methods Posterior longitudinal ligament cells were isolated from both OPLL patients and non-OPLL patients(n=5). The expression levels of miRNA-218 were compared between the 2 groups. Osteogenesis was induced after overexpressing miRNA-218 by agomir or inhibiting miRNA-218 by antagomir,in ligament cells of OPLL patients. The effect of miRNA-218 on the ossification of ligament cells was verified by Alizarin red staining,alkaline phosphatase activity and the expression of osteogenesis-related genes. Target-scan was used to predict the target gene of mi RNA-218,and dual-luciferase reporter assay was used to verify the targeting effect of microRNA-218. Results The expression level of miRNA-218 was significantly lower in OPLL patients than non-OPLL patients,and the difference was statistically significant(P < 0.05). The overexpression of miRNA-218 resulted in lower level of Alizarin red staining,alkaline phosphatase activities and expression of osteogenesis-related genes,while the inhibition resulted in higher level of Alizarin red staining,alkaline phosphatase activities and expression of osteogenesis-related genes,all with statistical significances(P < 0.05). Targetscan predicted that the target genes of miRNA-218 might be RUNX2 and typeⅠcollagen(COL1 A1). Dual-luciferase reporter assay showed that miRNA-218 could reduce the luciferase activity of RUNX2 and COL1 A1 genes. Conclusion mi RNA218 can significantly inhibit the ossification of primary posterior longitudinal ligament cells,and its mechanism is related to inhibiting the expression of RUNX2 and COL1 A1 genes.