Role of miR-494 and its target gene JunD in intervertebral disc herniation / 中华创伤杂志

ABSTRACT

Objective To investigate the expression and clinical implication of miR-494 and its target gene JunD in intervertebral disc herniation (IDH).Methods Six intervertebral disc tissue samples of spinal burst fracture were collected during operation,and then the nucleus pulposus cells were cultured.TNF-α of different concentration (0,10,50 and 100 ng/ml) were added to the cells to induce apoptosis.The apoptosis rate of nucleus pulposus cells and the expression of miR-494 were detected at different time after the stimulation (0,8,16 and 24 h) using qRT-PCR and flow cytometry respectively.Then,AntigomiR-494 was transfected into nucleus pulposus cells after lentivirus packaging,followed by the use of TNF-α (100 ng/ml,16 h) to induce apoptosis.The experiments contained blank control group,AntigomiR-494 + TNF-α group,and negative control + TNF-o group.Flow cytometry was used to detect the apoptosis in each group,and Western blot the expressions of JunD and cytochrome C.The luciferase double report based analysis and bioinformatics methods were used to investigate the relationship between miR-494 and JunD gene.Results The expression of miR-494 and the apoptosis rate of nucleus pulposus cells increased along with the increase of concentration of TNF-α and length of stimulation (P ＜ 0.05).After transfection,the expression of miR-494 and the apoptosis rate in AntigomiR-494 + TNF-α group were significantly lower than those in negative control + TNF-α Group (P ＜ 0.05).The results of Western blot showed that the expression level of JunD protein in AntigomiR-494 + TNF-α group was significantly higher than that of the negative control group (P ＜ 0.05),and the expression level of cytochrome C protein was significantly lower than that of the negative control + TNF-α group (P ＜ 0.05).Luciferase double report gene validation and bioinformatics prediction confirmed that miR-494 directly targeted JunD.Conclusion TNF-α can induce apoptosis of nucleus pulposus cells in a time and dose dependent manner.The expression of miR-494 increases with the increase of TNF-α concentration and length of stimulation.MiR-494 might be the key regulator of the apoptosis of nucleus pulposus cells under the induction of TNF-o.MiR-494 gene knockout can protect the nucleus pulposus cells,through the possible mechanism of upregulating target gene JunD and mediating the cytochrome C apoptosis pathway.MiR-494-JunD-cytochrome C signaling pathway might be one of the potential mechanisms of intervertebral disc degeneration.