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杨少锋,朱立国,李兆勇,李硕夫,郭彦涛,聂颖,张晨阳.miR-483/CREB1轴介导桃叶珊瑚苷抑制髓核细胞胞外基质降解的研究[J].湖南中医药大学学报英文版,2020,40(5):550-554.[Click to copy
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| miR-483/CREB1轴介导桃叶珊瑚苷抑制髓核细胞胞外基质降解的研究 |
| 杨少锋,朱立国,李兆勇,李硕夫,郭彦涛,聂颖,张晨阳 |
| (中国中医科学院望京医院, 北京 100102;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 研究miR-483/CREB1轴在桃叶珊瑚苷（aucubin，AU）抑制人退行性髓核（nucleus pulposus，NP）细胞胞外基质（extracellular matrix，ECM）降解中的功能作用。方法 AU处理人退行性NP细胞后，检测细胞活性、ECM相关蛋白及cAMP反应元件结合蛋白1（cAMP responsive element binding protein 1，CREB1）的表达。实时荧光定量PCR（quantitative real-time PCR，qPCR）和Western blot检测miR-483表达变化对CREB1丰度的影响；双荧光素酶报告实验（luciferase，LUC）检测miR-483和CREB1-3'-UTR的靶向结合；CREB1过表达载体和（或）miR-483 mimics共转染后，AU处理，检测CREB1及ECM相关蛋白的表达。结果 AU可显著增强NP细胞活性（P=0.000 4），抑制ECM降解酶基质金属蛋白酶-3（matrix metalloproteinase-3，MMP-3）、血小板反应蛋白解整合素金属肽酶-5（a disintegrin and metalloproteinase domain with thrombospondin motif-5，ADAMTS-5）与CREB1的表达，促进胶原蛋白Ⅱ型胶原α1（collagen type II alpha 1 chain，COL2A1）和miR-483的表达（P<0.001）。miR-483过表达可抑制CREB1的表达（P<0.000 1），LUC实验表明miR-483可与CREB1-3'-UTR靶向结合。功能实验结果表明CREB1可削弱AU对NP细胞ECM的降解的抑制作用，而miR-483可部分逆转CREB1对AU的抑制作用。结论 AU诱导NP细胞表达miR-483，进而抑制CREB1的表达，增强NP细胞的活性，抑制ECM的降解。 |
| 关键词: 桃叶珊瑚苷 髓核细胞 胞外基质 cAMP反应元件结合蛋白 微小RNA |
| DOI：10.3969/j.issn.1674-070X.2020.05.008 |
| Received:October 23, 2018 |
| 基金项目:湖南省自然科学基金项目（2017JJ2208）；湖南省教育厅科学研究项目（19C1392）。 |
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| Study on Aucubin-mediated MiR-483/CREB1 Axis Inhibits Extracellular Matrix Degradation in Human Degenerative Nucleus Pulposus Cells |
| YANG Shaofeng,ZHU Liguo,LI Zhaoyong,LI Shuofu,GUO Yantao,NIE Ying,ZHANG Chenyang |
| (Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China;The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To study the functional role of miR-483/CREB1 axis in the inhibition of extracellular matrix (ECM) degradation in human degenerative nucleus pulposus (NP) cells by aucubin (AU). Methods After treatment of human degenerative NP cells with AU, cell viability, ECM-related protein and cAMP responsive element binding protein 1 (CREB1) expression were measured. Quantitative real-time PCR (QPCR) and western blot were used to detect the effect of miR-483 expression on CREB1 abundance; dual luciferase reporter assay (LUC) was used to detect the targeted binding relationship between miR-483 and CREB1-3'-TUR; The CREB1 overexpression vector and/or miR-483 mimics were co-transfected into NP cells, and the expression of CREB1 and ECM-related proteins was detected after AU treatment. Results AU significantly enhanced the activity of NP cells (P=0.000 4), inhibited the expression of EMP degrading enzyme matrix metalloproteinase-3 (MMP-3) (P=0.000 3), a disintegrin and metalloproteinase domain with thrombospondin motif-5 (ADAMTS-5) and CREB1 (P<0.000 1), and promoted the expression of collagen type II alpha 1 chain (COL2A1) (P<0.000 1) and miR-483 (P=0.000 6). Overexpression of miR-483 inhibited the expression of CREB1 (P<0.000 1). LUC experiments indicated that miR-483 can bind to the CREB1-3'-UTR. Functional experiment showed that CREB1 can attenuate the inhibitory effect of AU on the degradation of ECM in NP cells, while miR-483 partially reverses the inhibitory effect of CREB1 on AU. Conclusion AU induces the expression of miR-483 in NP cells, thereby inhibiting the expression of CREB1, ultimately enhancing the activity of NP cells and inhibiting the degradation of ECM. |
| Key words: aucubin nucleus pulposus cells extracellular matrix cAMP responsive element binding protein 1 miRNA |
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