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
发布时间：2018-09-11 12:16
【摘要】：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(P0.05)。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(P0.05)。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(P0.05)。转染miR-4287模拟物后,软骨细胞内ADAMTS4 mRNA及蛋白表达均降低(P0.05);而转染miR-4287抑制物后,软骨细胞内ADAMTS4 mRNA及蛋白表达均升高(P0.05)。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(P0.05)。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。
[Abstract]:Objective to investigate the regulatory effect of chondrogenic miR-4287 on human chondrocyte polyproteoglycan enzyme 1 (a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) and its mechanism. Methods the normal and osteoarthritis cartilage tissues of knee joint were collected and the expression of miR-4287 and ADAMTS4 mRNA were detected by real-time fluorescence quantitative PCR, then the chondrocytes were isolated and cultured, and the first generation of osteoarthritis cells were collected and treated with IL-1 尾. The effects of SN50 on the expression of miR-4287 and ADAMTS4 mRNA in chondrocytes were observed, and the effects of IL-1 尾 -mediated signal pathway on miR-4287 and ADAMTS4 mRNA expression in chondrocytes were observed after pretreatment with MAPK signal pathway inhibitor SP600125 and NF- 魏 B signal pathway inhibitor SN50 with IL-1 尾 stimulation. The expression of ADAMTS4 mRNA and protein in chondrocytes was observed by transfection of miR-4287 analogue and its negative control inhibitor miR-4287 and negative control respectively. Luciferase reporting experiments demonstrated the direct binding effect of miR-4287 to ADAMTS4 mRNA 3 'untranslated region (untranslated region,UTR). Results compared with normal chondrocytes, the relative expression of miR-4287 in osteoarthritis cartilage decreased and the relative expression of ADAMTS4 mRNA increased. The difference was statistically significant (P0.05) .IL-1 尾 down-regulated miR-4287 expression in chondrocytes and up-regulated ADAMTS4 mRNA expression. Compared with the chondrocytes without IL-1 尾 treatment, the difference was statistically significant (P0.05). After pretreatment with signal pathway inhibitor mediated by IL-1 尾, the relative expression of miR-4287 in chondrocytes increased and the relative expression of ADAMTS4 mRNA in chondrocytes decreased, compared with the cells without signal pathway inhibitor pretreatment, the difference was statistically significant (P0.05). The expression of ADAMTS4 mRNA and protein in chondrocytes decreased after transfection of miR-4287 mimics (P0.05), while the expression of ADAMTS4 mRNA and protein in chondrocytes increased after transfection of miR-4287 inhibitors (P0.05). Overexpression of miR-4287 could not change the luciferase activity of the reporter vector, regardless of whether the binding site was wild type or mutant type (P0.05). Conclusion chondrogenic miR-4287 may be a kind of miRNA.miR-4287 associated with cartilage degeneration that can regulate the expression of ADAMTS4 in human chondrocytes, but not by targeting mRNA 3'UTR, and its specific mechanism needs further study.
【作者单位】： 中山大学附属第一医院关节外科;贵州医科大学附属医院骨科;
【基金】：国家自然科学基金资助项目(81572171) 广东省自然科学基金资助项目(2014A03031385)~~
【分类号】：R684.3
[Abstract]:Objective to investigate the regulatory effect of chondrogenic miR-4287 on human chondrocyte polyproteoglycan enzyme 1 (a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) and its mechanism. Methods the normal and osteoarthritis cartilage tissues of knee joint were collected and the expression of miR-4287 and ADAMTS4 mRNA were detected by real-time fluorescence quantitative PCR, then the chondrocytes were isolated and cultured, and the first generation of osteoarthritis cells were collected and treated with IL-1 尾. The effects of SN50 on the expression of miR-4287 and ADAMTS4 mRNA in chondrocytes were observed, and the effects of IL-1 尾 -mediated signal pathway on miR-4287 and ADAMTS4 mRNA expression in chondrocytes were observed after pretreatment with MAPK signal pathway inhibitor SP600125 and NF- 魏 B signal pathway inhibitor SN50 with IL-1 尾 stimulation. The expression of ADAMTS4 mRNA and protein in chondrocytes was observed by transfection of miR-4287 analogue and its negative control inhibitor miR-4287 and negative control respectively. Luciferase reporting experiments demonstrated the direct binding effect of miR-4287 to ADAMTS4 mRNA 3 'untranslated region (untranslated region,UTR). Results compared with normal chondrocytes, the relative expression of miR-4287 in osteoarthritis cartilage decreased and the relative expression of ADAMTS4 mRNA increased. The difference was statistically significant (P0.05) .IL-1 尾 down-regulated miR-4287 expression in chondrocytes and up-regulated ADAMTS4 mRNA expression. Compared with the chondrocytes without IL-1 尾 treatment, the difference was statistically significant (P0.05). After pretreatment with signal pathway inhibitor mediated by IL-1 尾, the relative expression of miR-4287 in chondrocytes increased and the relative expression of ADAMTS4 mRNA in chondrocytes decreased, compared with the cells without signal pathway inhibitor pretreatment, the difference was statistically significant (P0.05). The expression of ADAMTS4 mRNA and protein in chondrocytes decreased after transfection of miR-4287 mimics (P0.05), while the expression of ADAMTS4 mRNA and protein in chondrocytes increased after transfection of miR-4287 inhibitors (P0.05). Overexpression of miR-4287 could not change the luciferase activity of the reporter vector, regardless of whether the binding site was wild type or mutant type (P0.05). Conclusion chondrogenic miR-4287 may be a kind of miRNA.miR-4287 associated with cartilage degeneration that can regulate the expression of ADAMTS4 in human chondrocytes, but not by targeting mRNA 3'UTR, and its specific mechanism needs further study.
【作者单位】： 中山大学附属第一医院关节外科;贵州医科大学附属医院骨科;
【基金】：国家自然科学基金资助项目(81572171) 广东省自然科学基金资助项目(2014A03031385)~~
【分类号】：R684.3
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