天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (8): 871-875.doi: 10.11958/j.issn.0253-9896.2015.08.011

• 实验研究 • 上一篇    下一篇

白细胞介素-1β对大鼠软骨细胞MMP-13 表达的影响及 miR-27b 的调控作用

王欣，秦宇   

  1. 1天津医科大学（邮编300070）；2天津港口医院骨科
  • 收稿日期:2015-01-06 修回日期:2015-03-07 出版日期:2015-08-15 发布日期:2015-08-15
  • 作者简介:王欣（1979），男，大学本科，主治医师，主要从事骨科创伤研究

Effects of interleukin-1β on MMP-13 expression in rat chondrocytes and its regulation of miR-27b

WANG Xin, QIN Yu   

  1. 1 Tianjin Medical University, Tianjin 300070, China; 2 Department of Orthopedics, Tianjin Harbor Hospital
  • Received:2015-01-06 Revised:2015-03-07 Published:2015-08-15 Online:2015-08-15

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摘要： 目的观察白细胞介素（IL）-1β对大鼠软骨细胞基质金属蛋白酶（MMP）-13 表达的影响及miR-27b 的调控作用。方法雄性Wistar 大鼠7 只提取软骨细胞。Western blot 检测IL-1β刺激软骨细胞0 h、24 h、48 h 各时间点 MMP-13 表达变化；miRNAs 微阵列分析48 h 内软骨细胞差异表达的miRNAs；Real-time PCR 定量分析筛选出下调最为明显的miRNAs；荧光素酶报告基因实验验证miR-27b 与MMP-13 的靶定调控关系。结果IL-1β刺激软骨细胞后，MMP-13 蛋白在0 h、24 h、48 h 各时间点表达逐渐增加（P < 0.05）；miRNAs 微阵列分析发现48 h 内软骨细胞有36 个miRNAs 出现表达变化，变化最为明显的有6 个，分别为miR-27b、miR-31、miR-26a、miR-26b、miR-23、 miR-204；Real-time PCR 显示miR-27b 下调最为明显；miR-27b 拟似物和荧光素酶表达质粒共转染软骨细胞后，荧光素酶活性受到明显抑制（P < 0.05）。结论IL-1β刺激软骨细胞后，出现miR-27b 的表达下调和MMP-13 蛋白的表达上调，miR-27b 与MMP-13 存在靶定调控关系。

关键词: 软骨细胞, 基质金属蛋白酶13, 微RNAs, 白细胞介素1β, 微阵列分析, 微小RNA-27b

Abstract: Objective To observe the effect of interleukin-1β (IL-1β) on expression of Matrix Metalloproteinases 13 (MMP-13) in rat chondrocytes and its regulation of miR-27b. Methods Chondrocytes were extracted from 7 Wistar male rats. Expression of MMP-13 were examined by Western blot at 0 h, 24 h, 48 h after IL-1β stimulation. Differential miRNAs expression profiles were examined by miRNAs microarray. The most obviously down-regulated miRNAs were confirmed by quantitative Real-time PCR. Targeted regulation relationship between miR-27b and MMP-13 was set up by Luciferase re⁃ porter gene experiments. Results Expression of MMP-13 in rat chondrocytes was increased at a timely dependent manner upon IL-1β stimulation(P < 0.05)；Microarray revealed 36 miRNAs whose expression changed, among which 6（miR-27b, miR-31, miR-26a, miR-26b, miR-23, miR-204）were especially obvious. Real-time PCR confirmed that miR-27b was the one whose expression level were most down-regulated. Transient co-transfection of miR-27b mimics with luciferase expres⁃ sion plasmids resulted in significant repression of luciferase activity in rat chondrocytes (P < 0.05). Conclusion IL-1β stimulation result in down-regulation of miR-27b and up-regulation of MMP-13 expression. MiR-27b and MMP-13 show targeted regulation relationship.

Key words: chondrocytes, matrix metalloproteinase 13, microRNAs, interleukin-1beta, microarray analysis, miR-27b