title: MicroRNA-494 inhibition protects nucleus pulposus cells from TNF-α-induced apoptosis by targeting JunD.
abstract: BACKGROUND
  Human nucleus pulposus cell (HNPC) apoptosis plays an important role in the development of intervertebral disc degeneration (IVDD). Our previous research revealed that among all of the dysregulated microRNAs in the degenerated nucleus pulposus tissues of patient with IVDD, miRNA-494 (miR-494) is the most significantly increased. However, the influence of miR-494 HNPC apoptosis has not been confirmed.
  
  
  OBJECTIVE
  This study was designed to evaluate the effect of miR-494 on the HNPC apoptosis induced by TNF-α and to explore the possible mechanism of this process.
  
  
  METHODS
  First, 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 miR-494 expression. Second, we successfully knocked down endogenous miR-494 in HNPCs via lentiviral antigomiR-494 vector infection and then stimulated with TNF-α (100 ng/ml, 16 h). The rates of apoptosis and miR-494 expression were then detected again. Additionally, a dual-luciferase reporter assay and western blotting were used to determine whether JunD is a target of miR-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 miR-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 miR-494 expression were significantly decreased in the antigomiR-494+TNF-α group compared to the controls (p < 0.05). We also revealed that JunD is a target of miR-494. Western blotting analysis demonstrated that treatment with the lentiviral antigomiR-494 vector resulted in increased expression of JunD (p < 0.05) and decreased expression of cytochrome C (p < 0.05).
  
  
  CONCLUSIONS
  These results indicated that miR-494 is a novel regulator of HNPC apoptosis induced by TNF-α. The knock-out of miR-494 expression protected the HNPCs from apoptosis via the up-regulation of JunD, which was possibly mediated via cytochrome C apoptotic signaling. These findings suggest that the miR-494/JunD signaling pathway might represent a novel therapeutic target for the prevention of IVDD.
authors.affiliation: Tian Jin Medical University, No.22, Qixiangtai Road, Heping District, Tianjin 300070, China; Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Tian Jin Medical University, No.22, Qixiangtai Road, Heping District, Tianjin 300070, China; Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China. Electronic address: maxinlong86@163.com.
authors.forename: Xinlong
authors.initials: X
authors.lastname: Ma
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Tian
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Han
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Jiacheng
authors.initials: J
authors.lastname: Zang
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Jingbo
authors.initials: J
authors.lastname: Kong
authors.affiliation: Tian Jin Hospital, No.406, Jiefangnan Road, Hexi District, Tianjin 300211, China.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Yan
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology*; D008159:Lumbar Vertebrae / Q000166:cytology; D035683:MicroRNAs / Q000235:genetics*; D008928:Mitochondria / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D055815:Young Adult
chemical_list: C559371:JunD protein, human; C547168:MIRN494 microRNA, human; D035683:MicroRNAs; D016755:Proto-Oncogene Proteins c-jun; D014409:Tumor Necrosis Factor-alpha
keywords: Apoptosis; Intervertebral disc degeneration; JunD; MicroRNA; TNF-α