The Role Of MicroRNA-494 In TNF-α-induced Nucleus Pulposus Cell Apoptosis

Objective: The mechanism of molecular biology of intervertebral disc degeneration(IVDD) still keep magic. Human nucleus pulposus cell(HNPC) apoptosis plays an important role in the development of intervertebral disc degeneration(IVDD). TNF-acan induce nucleus pulposus cells apoptosis, which has an intimate relationship with IVDD. The extrinsic factor can combine with cell surface receptor, and produce biological effect through endogenous factor, for example mi RNAs. Our previous research revealed that among all of the dysregulated micro RNAs in the degenerated nucleus pulposus tissues of patient with IVDD, mi R-494(mi R-494) is the most significantly increased. However, the influence of mi R-494 on TNF-a-induced HNPC apoptosis has not been confirmed. This study was designed to evaluate the effect of mi R-494 on the HNPC apoptosis induced by TNF-α and to explore the possible mechanism of this process.Methods: Collect the clinical data and nucleus pulposus of spine burst fracture patients. According to preoperative imaging data(X-ray, CT, MRI), we evaluate the situation of patients by TLISS score system and load sharing scores. According to the MRI, the degree of degeneration of IVDD is evaluated by Pfirrmann classification.We used enzyme digestion method to obtain primary nucleus pulposus cells, and appraised nucleus pulposus cells. HNPCs were stimulated with TNF-α at different concentrations(0 ng/ml, 10 ng/ml, 50 ng/ml, or 100 ng/ml) for 0 h, 8 h, 16 h, or 24 h.Annexin V-PE/7-AAD assays and real-time quantitative PCR were used to detect the cell apoptosis rates and mi R-494 expression. Second, we successfully knocked down endogenous mi R-494 in HNPCs via lentiviral antigomi R-494 vector infection and then stimulated with TNF-α(100 ng/ml, 16 h). The rates of apoptosis and mi R-494 expression were then detected again. Additionally, a dual-luciferase reporter assay and western blotting were used to determine whether Jun D is a target of mi R-494.Finally, western blotting was used to analyze the expression of cytochrome C.Results: We found that the rate of apoptosis increased with concentration, time(p<0.05) and mi R-494 expression(p<0.05). The rate of apoptosis in the 100 ng/ml, 16 h group appeared to be suitable. After transfection, the apoptosis rate and mi R-494expression were significantly decreased in the antigomi R-494+TNF- a group compared to the controls(P<0.05). We also revealed that Jun D is a target of mi R-494.Western blotting analysis demonstrated that treatment with the lentiviral antigomi R-494 vector resulted in increased expression of Jun D(P<0.05) and decreased expression of cytochrome C(P<0.05).Conclusion: These results indicated that mi R-494 is a novel regulator of HNPC apoptosis induced by TNF-α. The knock-out of mi R-494 expression protected the HNPCs from apoptosis via the up-regulation of Jun D, which was possibly mediated via cytochrome C apoptotic signaling. These findings suggest that the mi R-494/Jun D signaling pathway might represent a novel therapeutic target for the prevention of IVDD.