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

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(c AMP responsive element binding protein 1,CREB1)的表达。实时荧光定量PCR(quantitative real-time PCR,q PCR)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=0.0004),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<0.0001),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。

Study on Aucubin-mediated MiR-483/CREB1 Axis Inhibits Extracellular Matrix Degradation in Human Degenerative Nucleus Pulposus Cells

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=0.0004),inhibited the expression of EMP degrading enzyme matrix metalloproteinase-3(MMP-3)(P=0.0003),a disintegrin and metalloproteinase domain with thrombospondin motif-5(ADAMTS-5)and CREB1(P<0.0001),and promoted the expression of collagen type II alpha 1 chain(COL2 A1)(P<0.0001)and miR-483(P=0.0006).Overexpression of miR-483 inhibited the expression of CREB1(P<0.0001).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.