【摘要】 目的 探讨microRNA-495(miR-495)对人血管内皮细胞增殖的作用及其分子作用机制。方法 选择人脐静脉内皮细胞(HUVECs),利用lipofectamine2000转染试剂,将miR-495 mimics(模拟物)或miR-495 inhibitors(抑制物)转染HUVECs,分别以mimics control和inhibitors control作为对照。通过MTT实验分析过表达或干扰miR-495对HUVECs细胞增殖的影响,进而通过生物信息学软件预测,双荧光素酶报告基因系统,荧光定量PCR及Western blot,预测并验证miR-495靶基因,最后通过功能互补实验,观察提高或敲低增殖细胞核抗原(PCNA)表达对HUVECs细胞增殖的影响。结果 体外细胞增殖实验发现,过表达miR-495可明显抑制HUVECs细胞的增殖,而干扰miR-495则可促进HUVECs细胞的增殖。PCNA是miR-495的靶基因,过表达miR-495可明显下调HUVECs细胞中PCNA mRNA及蛋白表达水平。功能互补实验显示,敲低PCNA的表达亦可降低HUVECs细胞的增殖,反之促进HUVECs细胞的增殖。结论 MiR-495可抑制HUVECs细胞的增殖,其作用机制主要通过下调PCNA的表达而实现。更多还原

【Abstract】 Objective To investigate MicroRNA-495(miR-495) inhibits human vascular endothelial cells proliferation,and clarify its molecular mechanism.Methods We applied human umbilical vein endothelial cells(HUVECs) and cultured in Dulbecco’s modified Eagles mediu(DMEM).MTT assay was used to explore the proliferation ability of HUVECs,after transfected with miR-495 mimics or miR-495 inhibitors,with mimics control or inhibitors control as experimental control.Bioinformaties,luciferase reporter assay,Quantitative RT-PCR and Western blot analyses were used to predict and verify miR-495 target in HUVECs.Finally,HUVECs proliferation was also detected by using MTT assay after knock-down or over-expression PCNA(proliferating cell nuclear antigen).Results Over-expression of miR-495 could significantly inhibit HUVECs proliferation,while interfering miR-495 expression could promote its proliferation in vitro.PCNA was a target gene of miR-495 in HUVECs and its mRNA and protein expression levels were significantly decreased after transfected with miR-495 mimics.Functions complementary experiments showed that knock-down PCNA could also arrest the HUVECs proliferation,and over-expression PCNA promoted cells proliferation.Conclusion MiR-495 could inhibit HUVECs cells proliferation,and its mechanism of action mainly executed by suppressing expression of PCNA.更多还原