[MicroRNA-563 promotes the osteogenic differentiation of posterior longitudinal ligament cells by inhibiting SMURF1].

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.