title: [MicroRNA-563 promotes the osteogenic differentiation of posterior longitudinal ligament cells by inhibiting SMURF1].
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 byttest.Results:The expression level of miR-563 was significantly higher in OPLL than PLL groups (8.53±0.84vs. 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±0.25vs.1.00±0.14), alkaline phosphatase activities (3.11±0.55vs.1.00±0.11) and ossification related genes (RUNX2: 3.25±0.55vs.1.00±0.10; IBSP: 2.35±0.32vs. 1.00±0.14;t:7.43 to 10.99, allP=0.000), while the inhibition resulted in lower level (alizarin red staining: 0.52±0.21vs. 1.00±0.12; alkaline phosphatase activities: 0.41±0.12vs. 1.00±0.09; RUNX2: 0.35±0.13vs. 1.00±0.12; IBSP: 0.55±0.12vs.1.00±0.11;t:4.36 to 8.45, allP<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±0.06vs.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 cellsin vitro,and the mechanism of which is possibly through down regulating SMURF1.
authors.affiliation: First Department of Spinal Surgery, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai 200003, China.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: C
authors.initials: C
authors.lastname: Xu
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: W
authors.initials: W
authors.lastname: Yuan
mesh_terms: D000469:Alkaline Phosphatase; D002454:Cell Differentiation; D006801:Humans; D017846:Longitudinal Ligaments; D035683:MicroRNAs; D017887:Ossification of Posterior Longitudinal Ligament; D010012:Osteogenesis; D044767:Ubiquitin-Protein Ligases
chemical_list: D035683:MicroRNAs; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D000469:Alkaline Phosphatase
keywords: Gene expression regulation; MicroRNA; Ossification of posterior longitudinal ligament; Osteogenesis