miR-21/PTEN/Akt参与白藜芦醇促EPC的体外成血管能力

Effects of miR-21/PTEN/Akt signal pathway on the angiogenesis of EPC promoted by resveratrol

目的探讨miR-21及其下游PTEN/Akt信号通路在白藜芦醇促进大鼠骨髓源性内皮祖细胞(EPC)体外成血管能力中的作用。方法采用密度梯度离心法分离大鼠四肢骨髓的单个核细胞,培养于含10%胎牛血清的EGM-2MV完全培养基中诱导分化成EPC,实验用3~5代的EPC。白藜芦醇(20μmol/L)干预12 h后,采用Matrigel胶检测EPC的体外成血管能力;实时荧光定量PCR检测miR-21及PTEN基因的表达情况;干扰miR-21表达后检测EPC的体外成血管能力;双荧光素酶报告基因检测miR-21对PTEN的靶向调控;Western blot检测PTEN蛋白表达情况以及Akt磷酸化水平。结果白藜芦醇(20μmol/L)明显促进EPC的体外成血管能力(P<0.05),抑制了miR-21(P<0.01)的表达,然而却促进了miR-21下游靶基因PTEN的基因(P<0.01)及蛋白表达(P<0.05),进而抑制PTEN下游信号分子Akt的磷酸化水平。双荧光素酶报告基因检测结果显示,miR-21可与PTEN mRNA的3′UTR靶向结合(P<0.01)。结论miR-21调控了白藜芦醇促EPC的体外成血管能力,其机制可能是通过PTEN/Akt信号通路来发挥的,该研究结果揭示了白藜芦醇调控EPC功能一种新的细胞内信号机制。

Aim To investigate the role of miR-21/PTEN/Akt signal pathway in resveratrol promoting angiogenesis of endothelial progenitor cells(EPC)isolated from rat bone marrow.Methods Mononuclear cells were isolated by density gradient centrifugation from rat bone marrow and then cultured with EGM-2MV containing 10%fetal bovine serum.3~5 generations of EPC were used in the experiments.EPC were treated with resveratrol(20μmol/L)for 12 h.And then matrigel,real-time PCR and Western blot were used to detect the angiogenesis,the expression of miR-21 and PTEN mRNA,and the PTEN protein and Akt phosphorylation level of EPC in vitro separately.The dual luciferase reporter system was employed to detect the binding of miR-21 and PTEN mRNA.Results Resveratrol(20μmol/L)significantly promoted the angiogenesis of EPC in vitro(P<0.05).In the meantime,resveratrol inhibited the expression of miR-21 which promoted the gene and protein expression of PTEN.PTEN furtherly inhibited the phosphorylation level of Akt.The results of the double luciferase reporter system showed that miR-21 bound to the 3′UTR of PTEN mRNA.Conclusion miR-21 regulated the angiogenesis ability of EPC promoted by resveratrol.The present study indicated that miR-21/PTEN/Akt signaling pathway may play an important role in EPC angiogenesis promoted by resveratrol in vitro.