摘要
目的:探讨微小核糖核酸(microRNA)-24(miR-24)对内皮型一氧化氮合酶(eNOS)基因表达调节的分子机制及其对血管内皮细胞增殖、迁移和管腔形成能力的影响。方法:构建miR-24及其反义序列的高表达质粒,分别转染人脐静脉内皮细胞(HUVECs),根据转染质粒将实验细胞分为:miR-24高表达组、miR-24干扰组和空白质粒对照组。用四甲基偶氮唑盐(MTT)检测HUVECs增殖能力,划痕和Transwell试验检测细胞的迁移能力,人工基底膜检测细胞的管腔形成能力;分别用逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blotting)检测eNOS和Sp1转录因子m RNA和蛋白表达水平。结果:(1)与空白质粒对照组比较,miR-24高表达组细胞增殖能力降低45.45%(0.36±0.04 vs 0.66±0.08,P<0.05);miR-24高表达组细胞迁移速度明显减缓,且迁移数目降低74.75%(30.25±3.78 vs 119.80±10.94,P<0.01),未能形成明显管腔样结构。(2)与空白质粒对照组比,miR-24高表达组eNOS m RNA降低46.2%(0.49±0.02vs 0.91±0.01,P<0.05),蛋白表达减少49.07%(0.55±0.05 vs 1.08±0.05,P<0.05);同时Sp1 m RNA降低44.9%(0.49±0.01 vs 0.89±0.02,P<0.05),其蛋白质表达量也相应减少54.90%(0.46±0.02 vs 1.02±0.04,P<0.05)。在miR-24抑制组中,上述指标较空白质粒对照组降低,但比miR-24高表达组显著升高,特别是小管形成数量、及管腔长度与空白质粒对照组相近。结论:miR-24显著抑制HUVECs的增殖、迁移和管腔形成的能力,并且与miR-24对eNOS的表达调控有关;miR-24明显抑制eNOS表达,Sp1的参与可能是这一调节过程的重要分子机制之一。
Objective： To investigate the effects of miR-24 on endothelial nitric oxide synthase （eNOS） gene expression with regulation and endothelial cell proliferation, migration, tube formation in human umbilical vein endothelial cells （HUVECs）. Methods： Constructed high expression plasmid of miR-24 and miR-24 antisense sequence were introduced into HUVECs and the cells included in 3 groups： Control group, miR-24 group and miR-24 inhibitor group. HUVEC proliferation was detected by MTT test, migration was measured by Scratching and Transwell methods, tube formation was examined by Matrigel assay; mRNA and protein expressions of eNOS and Sp1were determined by RT-PCR and Western blot analysis respectively. Results：①Compared with Control group, miR-24 group had decreased cell proliferation by 45.45% as （0.36 ± 0.04） vs （0.66 ± 0.08）,P〈0.05; miR-24 group had lower speed of cell migration, decreased number of cell migration by 74.75% as （30.25±3.78） vs （119.80±10.94）,P〈0.01 and there was no obvious tube formation.②Compared with Control group, miR-24 group showed reduced eNOS mRNA expression by 46.2% as （0.49±0.02） vs （0.91±0.01）,P〈0.05, reduced protein expression by 49.07% as （0.55±0.05） vs （1.08±0.05）,P〈0.05; meanwhile, decreased Sp1 mRNA expression by 44.9% as （0.49±0. 01） vs （0. 89±0.02）P〈0.05, decreased protein expression by 54.90% as （0.46±0.02） vs （1.02±0.04）,P〈0.05. In miR-24 inhibitor group, the above indexes were lower than Control group but higher than miR-24 group, the amount of tube formation and the length of tubes were similar between Control group and miR-24 inhibitor group. Conclusion： MiR-24 may inhibit HUVECs proliferation, migration, tube formation and suppress eNOS expression; Sp1 might be one of the important regulators.
出处
《中国循环杂志》
CSCD
北大核心
2016年第8期797-801,共5页
Chinese Circulation Journal
基金
国家自然科学基金项目(81373403)
广西研究生科研创新项目(YCSZ2015119)
关键词
核糖核酸
管腔
一氧化氮合酶
内皮型
RNA
Tube formation
Endothelial nitric oxide synthase
