miR-494及其靶基因JunD在损伤疝出型椎间盘突出中的表达及其临床意义  

Role of miR-494 and its target gene JunD in intervertebral disc herniation

作　　者：王涛[1] 马信龙[1] 李鹏飞[1] 韩超[1] 田鹏[1] Wang Tao, Ma Xinlong, Li Pengefei, Han Chao, Tian Peng.(Department of Spine Surgery, Tianjin Hospital, Tianjin 300211, Chin)

机构地区：[1]天津医院脊柱外科,天津300211

出　　处：《中华创伤杂志》2018年第5期457-464,共8页Chinese Journal of Trauma

基　　金：天津市卫生行业重点攻关项目基金（16KG140）

摘　　要：目的 探讨miR-494及其靶基因JunD在损伤疝出型椎间盘突出（IDH）中的表达及其临床意义. 方法 收集脊柱爆裂骨折手术椎间盘标本6个,髓核细胞培养,不同浓度（0,10,50,100 ng/ml）肿瘤坏死因子-α（TNF-α）诱导髓核细胞凋亡,建立髓核细胞凋亡模型,于不同时相点（0,8,16,24 h）采用qRT-PCR和流式细胞术检测髓核细胞凋亡率及miR-494的表达.AntigomiR-494在慢病毒包装后转染髓核细胞,成功敲除髓核细胞miR-494后使用TNF-α（100 ng/ml,16 h）诱导髓核细胞凋亡.采用随机数字表法进行实验分组：空白对照组（髓核细胞）、AntigomiR-494＋ TNF-α组、阴性对照＋TNF-α组.采用流式细胞术检测各组细胞凋亡情况.Western blot检测JunD蛋白和细胞色素C蛋白的表达情况.荧光素酶双报告基因分析技术及生物信息学方法了解miR-494与JunD基因的靶向关系. 结果 miR-494表达量及髓核细胞凋亡率随TNF-α浓度和刺激时间的增加而增加（P＜0.05）.与阴性对照＋TNF-α组相比,在细胞转染后,AntigomiR-494＋ TNF-α组miR-494表达量和细胞凋亡率明显下降（P＜0.05）.Westernblot结果表明,AntigomiR-494＋ TNF-α组JunD蛋白的表达水平较阴性对照＋TNF-α组高（P＜0.05）,而细胞色素C蛋白的表达水平较阴性对照＋TNF-α组低（P＜0.05）.荧光素酶双报告基因验证、生物信息学方法预测证实miR-494直接靶向JunD. 结论 TNF-α可诱导髓核细胞的凋亡,且呈时间和剂量依赖性.髓核细胞miR-494的表达量随TNF-α的刺激浓度、时间增加而增加.miR-494有可能是髓核细胞在TNF-α诱导下凋亡的关键调节器.miR-494基因敲除可以保护髓核细胞,其可能机制是上调靶基因JunD,并且介导细胞色素C凋亡通路.miR-494-JunD-细胞色素C信号通路可能是椎间盘退变的潜在机制之一.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

关 键 词：椎间盘移位 肿瘤坏死因子Α JUND 

分 类 号：R681.53[医药卫生—骨科学]