[MicroRNA-563 promotes the osteogenic differentiation of posterior longitudinal ligament cells by inhibiting SMURF1].
[MicroRNA-563 promotes the osteogenic differentiation of posterior longitudinal ligament cells by inhibiting SMURF1].
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DOI：
10.3760/cma.j.issn.0529-5815.2017.03.008
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发表时间：
2017-03
期刊：
影响因子：
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通讯作者：
H. Zhang;C. Xu;Y. Liu;W. Yuan
中科院分区：
文献类型：
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作者：
H. Zhang;C. Xu;Y. Liu;W. Yuan
目的：探讨miR-563对后纵韧带（OPLL）细胞骨化的调控作用及其机制。研究方法：方法：收集2015年3月至2015年6月在第二军医大学附属长征医院脊柱外科一科行脊柱手术的OPLL患者（n=6）和非骨化韧带患者（PLL，n=4）的后纵韧带细胞进行分离培养。采用实时荧光定量PCR分析OPLL和PLL组中miR-563的表达水平。利用microRNA模拟物来过表达miR-563，并设计microRNA抑制剂来敲低其表达。采用过表达和抑制法，分析miR-563过表达或抑制及骨化诱导韧带细胞中茜素红染色、碱性磷酸酶和骨化相关基因的水平。然后利用Targetscan预测miR-563的潜在靶点，并利用双荧光素酶报告基因检测进行验证。组间比较采用t检验。结果：OPLL组miR-563的表达水平明显高于PLL组（8.53± 0.84vs.1.00 ±0.12，t '= 21.629，P=0.000）。miR-563的过表达导致茜素红染色水平升高（2.52±0.25 vs.1.00±0.14），碱性磷酸酶活性（3.11±0.55 vs.1.00±0.11）和骨化相关基因（RUNX 2：3.25±0.55 vs.1.00±0.10; IBSP：2.35±0.32 vs.1.00 ±0.14; t：7.43 ~ 10.99，均P=0.000），而抑制则导致较低水平的（茜素红染色：0.52±0.21 vs. 1.00±0.12;碱性磷酸酶活性：0.41±0.12 vs. 1.00±0.09; RUNX 2：0.35±0.13 vs. 1.00±0.12; IBSP：0.55±0.12 vs.1.00±0.11; t：4.36 ~ 8.45，均P<0.05）。结合Targetscan的预测结果和OPLL和PLL的表达谱，发现SMURF 1是miR-563的潜在靶点，双荧光素酶报告基因分析也证实了它们之间的关系。SMURF 1过表达后，SMURF 1的表达水平显著降低（0.25±0.06 vs.1.00±0.10，t=-12.862，P =0.000），再次验证了上述假设。结论：miRNA-563在体外能显著促进后纵韧带细胞的成骨分化，其机制可能与下调SMURF 1有关。
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.