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Title: [MicroRNA-563 promotes the osteogenic differentiation of posterior longitudinal ligament cells by inhibiting SMURF1].
Author: Zhang H, Xu C, Liu Y, Yuan W.
Journal: Zhonghua Wai Ke Za Zhi; 2017 Mar 01; 55(3):203-207. PubMed ID: 28241722.
Abstract:
Objective: To investigate the function and mechanism of miR-563 in regulating the ossification of posterior longitudinal ligament (OPLL) cells. Methods: Posterior longitudinal ligament cells were isolated and cultured from both OPLL patients (n=6) and non-ossified ligament patients (PLL, n=4) who underwent spine surgery from March to June 2015 in First Department of Spinal Surgery, Changzheng Hospital Affiliated to Second Military Medical University. The expression levels of miR-563 in OPLL and PLL groups were analyzed using real-time PCR. MicroRNA mimics were utilized to over express miR-563, and microRNA inhibitors were designed to knockdown its expression. Using the over expression and inhibition method, the level of Alizarin Red staining, alkaline phosphatase and ossification related genes in miR-563 were analyzed over expressed or inhibited and ossification induced ligament cells. After that the potential target of miR-563 was predicted using Targetscan and verified using dual-luciferase reporter assay. The results between the groups were compared by t test. Results: The expression level of miR-563 was significantly higher in OPLL than PLL groups (8.53±0.84 vs. 1.00±0.12, t'=21.629, P=0.000). The over expression of miR-563 resulted in higher level of alizarin red staining (2.52±0.25 vs.1.00±0.14), alkaline phosphatase activities (3.11±0.55 vs.1.00±0.11) and ossification related genes (RUNX2: 3.25±0.55 vs.1.00±0.10; IBSP: 2.35±0.32 vs. 1.00±0.14; t: 7.43 to 10.99, all P=0.000), while the inhibition resulted in lower level (alizarin red staining: 0.52±0.21 vs. 1.00±0.12; alkaline phosphatase activities: 0.41±0.12 vs. 1.00±0.09; RUNX2: 0.35±0.13 vs. 1.00±0.12; IBSP: 0.55±0.12 vs.1.00±0.11; t: 4.36 to 8.45, all P<0.05). Combining the prediction results of Targetscan and expression profiles between OPLL and PLL, SMURF1 was found as a potential target of miR-563, and dual-luciferase reporter assay also identified their relationship. By over expression, the expression level of SMURF1 was significantly decreased (0.25±0.06 vs.1.00±0.10, t=-12.862, P=0.000), which again verified the hypothesis. Conclusion: miRNA-563 significantly promotes the osteogenic differentiation of posterior longitudinal ligament cells in vitro, and the mechanism of which is possibly through down regulating SMURF1. 目的：探讨后纵韧带骨化(OPLL)特异性微小RNA-563(miR-563)在OPLL患者后纵韧带细胞成骨分化中的作用及机制。 方法：选择2015年3月至6月在第二军医大学长征医院脊柱外科接受手术治疗的6例OPLL患者，于颈椎前路减压手术中取OPLL患者未骨化的后纵韧带部分(OPLL组)，选择同期在同一单位接受手术治疗的4例颈椎外伤患者，以同样方法于术中取其后纵韧带(PLL组)，采用贴壁法对获取的后纵韧带进行体外培养，利用real-time PCR检测两组间miR-563的表达差异。对OPLL组后纵韧带细胞转染RNA oligo过表达和抑制miR-563后进行成骨诱导，通过检测茜素红染色水平、碱性磷酸酶水平及成骨相关基因的表达验证miR-563在OPLL组后纵韧带细胞成骨分化中的作用。预测miR-563靶基因，并利用双荧光素酶报告基因实验验证其靶向作用。应用t检验对组间的数据进行比较。 结果： OPLL组miR-563的表达水平高于PLL组(8.53±0.84比1.00±0.12，t′＝21.629，P＝0.000)。韧带细胞过表达miR-563后茜素红染色水平(2.52±0.25比1.00±0.14)、碱性磷酸酶水平(3.11±0.55比1.00±0.11)及成骨相关基因表达(RUNX2：3.25±0.55比1.00±0.10；IBSP：2.35±0.32比1.00±0.14)均高于对照组(t值为7.43～10.99，P值均＝0.000)。而抑制miR-563后则上述指标较对照组出现明显下调(茜素红染色：0.52±0.21比1.00±0.12；碱性磷酸酶：0.41±0.12比1.00±0.09；RUNX2：0.35±0.13比1.00±0.12；IBSP：0.55±0.12比1.00±0.11；t值为－8.45～－4.36，P值均<0.05)。通过Targetscan预测miR-563的靶基因为SMURF1，通过过表达miR-563检测SMURF1表达水平发生明显下调(0.25±0.06比1.00±0.10，t＝－12.862，P＝0.000)，双荧光素酶报告基因检测明确了其靶向机制(0.22±0.03比1.00±0.09，t＝－16.444，P＝0.000)。 结论： miR-563能够明显促进OPLL患者后纵韧带原代细胞的成骨分化，其作用与靶向抑制SMURF1相关。.

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