中国修复重建外科杂志

中国修复重建外科杂志

成软骨相关 miR-4287 调控人软骨细胞聚蛋白多糖酶-1 表达的研究

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目的 探讨成软骨相关 miR-4287 对人软骨细胞聚蛋白多糖酶-1(a disintegrin and metalloproteinase with thrombospondin motif 4,ADAMTS4)调控作用及其机制。 方法 取自愿捐赠的膝关节正常及骨关节炎软骨组织,采用实时荧光定量 PCR 检测 miR-4287 和 ADAMTS4 mRNA 表达量;然后分离培养软骨细胞,取第 1 代骨关节炎细胞,给予 IL-1β 处理,观察其对软骨细胞 miR-4287 和 ADAMTS4 mRNA 表达的影响;分别给予 MAPK 信号通路抑制剂 SP600125 以及 NF-κB 信号通路抑制剂 SN50 预处理后联合 IL-1β 刺激,观察 IL-1β 介导的信号通路对软骨细胞 miR-4287 和 ADAMTS4 mRNA 表达的影响;分别转染 miR-4287 模拟物及其阴性对照、miR-4287 抑制物及其阴性对照,观察 miR-4287 调控软骨细胞 ADAMTS4 mRNA 及蛋白的表达。荧光素酶报告实验验证 miR-4287 与 ADAMTS4 mRNA 3’非翻译区(untranslated region,UTR)的直接结合效应。 结果 与正常软骨组织比较,骨关节炎软骨组织 miR-4287 相对表达量下降,ADAMTS4 mRNA 相对表达量上升,比较差异有统计学意义(P<0.05)。IL-1β 下调软骨细胞 miR-4287 表达、上调 ADAMTS4 mRNA 表达,与未经 IL-1β 处理的软骨细胞相比差异均有统计学意义(P<0.05)。经 IL-1β 介导的信号通路抑制剂预处理后,软骨细胞 miR-4287 相对表达量上升,ADAMTS4 mRNA 相对表达量降低,与未经信号通路抑制剂预处理细胞相比,差异均有统计学意义(P<0.05)。转染 miR-4287 模拟物后,软骨细胞内 ADAMTS4 mRNA 及蛋白表达均降低(P<0.05);而转染 miR-4287 抑制物后,软骨细胞内 ADAMTS4 mRNA 及蛋白表达均升高(P<0.05)。无论结合位点为野生型或突变型,过表达 miR-4287 均不能改变报告载体的荧光素酶活性(P>0.05)。 结论 成软骨相关 miR-4287 可能是一种与软骨退变相关的 miRNA。miR-4287 能调控人软骨细胞 ADAMTS4 表达,但不是通过靶向结合 mRNA 3’UTR 的方式发挥作用,其具体机制有待进一步研究。

Objective To investigate the effect and mechanism of miR-4287, a chondrogenesis associated microRNA, regulated the expression of aggrecanase-1 (a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4) in human chondrocytes. Methods First, the voluntarily donated normal and osteoarthritic knee articular cartilages were used to detect the expressions of miR-4287 and ADAMTS4 mRNA by real-time fluorescence quantitative PCR. Then, chondrocytes were isolated from knee articular cartilages. The effect of IL-1β on the expression of miR-4287 and ADAMTS4 mRNA was validated by the first generation of osteoarthritic chondrocytes. To confirm the influence of IL-1β signal pathways on the expression of miR-4287 and ADAMTS4 mRNA, osteoarthritic chondrocytes were pretreated with MAPK signal pathway inhibitor SP600125, NF-κB pathway inhibitor SN50, and finally stimulated with IL-1β. Chondro cytes were transfected with miR-4287 mimics and mimics negative control, inhibitors and inhibitors negative control respectively to value the effect of miR-4287 on ADAMTS4 expression. Luciferase reporter assay was used to verify the direct interaction between miR-4287 and putative site in the 3-untranslated region (3’UTR) of ADAMTS4 mRNA. Results Compared with normal knee articular cartilages, the miR-4287 expression was markedly diminished and conversely ADAMTS4 mRNA expression was raised in osteoarthritis cartilages (P<0.05). Stimulation with IL-1β led to a reduction in miR-4287 expression and upregulation in ADAMTS4 mRNA expression, showing significant difference when compared with the untreated groups (P<0.05). Pretreatment with IL-1β signal pathway inhibitors induced miR-4287 expression and attenuated ADAMTS4 mRNA expression in human chondrocytes, which were significantly different from that of unstimulated cells (P<0.05). ADAMTS4 mRNA and protein were suppressed by transfection with miR-4287 mimics (P<0.05) and elevated by transfection with miR-4287 inhibitors (P<0.05). As luciferase reporter assay showed, overexpression miR-4287 failed to alter the luciferase activity of a reporter construct containing either wild or mutant 3’UTR of ADAMTS4 mRNA (P>0.05). Conclusion miR-4287, a chondrogenesis associated microRNA, may play an important role in cartilage degeneration. miRNA-4287 is able to regulate ADAMTS4 expression in human chondrocytes, but not by means of directly targeted the ADAMTS4 mRNA 3’UTR. The exact mechanisms need to be further addressed.

关键词: 骨关节炎; miR-4287; IL-1β; 聚蛋白多糖酶-1; 软骨细胞

Key words: Osteoarthritis; miR-4287; interleukin 1β; aggrecanase-1; chondrocytes

引用本文: 孙红, 张志奇, 黄志宇, 毛谷平, 余宝禧, 张程云, 傅明. 成软骨相关 miR-4287 调控人软骨细胞聚蛋白多糖酶-1 表达的研究. 中国修复重建外科杂志, 2017, 31(12): 1468-1473. doi: 10.7507/1002-1892.201704065 复制

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