内含子源性27nt-miRNA对内皮型一氧化氮合酶表达调节及血管内皮细胞管腔形成的影响被引量：2

INTRONIC 27NT-MIRNA REGULATES EXPRESSION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE AND TUBE TORMATION OF VASCULAR ENDOTHELIAL CELL

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摘要目的:探讨内含子源性27nt-miRNA对内皮型一氧化氮合酶(eNOS)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wt-27nt-miRNA、mut-27nt-miRNA和空白对照序列的高表达质粒,分别转染人脐静脉血管内皮细胞(HUVECs),采用RT-PCR、Western blotting检测eNOS mRNA和蛋白表达水平;MTT检测HUVECs增殖能力,划痕和Transwell实验检测HUVECs的迁移能力,Matrigel检测HUVECs的管腔形成能力。结果:(1)与空白质粒对照组比较,wt-27nt-miRNA表达组HUVECs eNOS mRNA降低39.51%(0.49±0.02vs 0.81±0.02,P<0.05),其蛋白质表达量下降47.41%(0.71±0.07vs 1.35±0.06,P<0.05)。(2)与空白质粒对照组相比,wt-27nt-miRNA表达组HUVECs增殖明显减少,抑制率为41.86%;wt-27nt-miRNA表达组HUVECs迁移速度明显减缓,且迁移数目降低75.55%(28.75±1.71vs117.60±4.45,P<0.01),未能形成明显管腔样结构。在mut-27nt-miRNA表达组中,上述指标比空白质粒对照组降低(P<0.05),但比wt-27nt-miRNA表达组显著升高(P<0.05),特别是管腔形成数量及管腔长度与空白质粒对照组相近。结论:内含子源性27nt-miRNA显著抑制HUVECs的增殖、迁移和管腔形成的能力,并与内含子源性27nt-miRNA对eNOS表达的调控相关。 Objective： This study was to investigate the effect of intronic 27nt-miRNA on eNOS expression and the capacities of endothelial cell proliferation, migration and tube formation. Methods： We constructed high expression plasmids of wt-27nt-miRNA, mut-27nt-miRNA and introduced control sequence into HU- VECs. The expressions of eNOS at mRNA and protein levels were examined by real-time PCR and West- ern blotting. The HUVECs proliferation was detected by MTT assay. Scratch assay and Transwell assay were used to measure the HUVECs migration. Tube formation was detected by Matrigel assay. Results： Compared with control group, the expression of eNOS at mRNA and protein levels was decreased by 39.51%（0.49±0.02 vs 0.81±0.02, P 〈0.05）and by 47.41%（0.71±0.07 vs 1.35±0.06, P 《0.05）; and the proliferation of HUVECs in wt-27nt-miRNA group was significantly decreased, with an inhibition ratio of 41.86 %. The HUVECs motility was slower than that in the control group, and the migrated HU- VECs in wt-27nt-miRNA group were significantly decreased by 75.55%（28.75±1.71 vs 117.60±4.45, P 〈0.01）. Matrigel assay showed few capillaries. In mut-27nt-miRNA group, the above indexes were decreased compared with control group（ P 〈0.05）, but significantly increased compared with wt-27nt-miR- NA group（ P 〈0.05）, especially the number of branches and the length of the tubes formed were similar to those in the control group. Conclusion： Intronic 27nt-miRNA significantly suppresses cell proliferation,migration and tube formation of HUVECs, which might be associated with regulation of eNOS.

作者陈伟莫国君罗雪兰杨鹏欧和生

机构地区广西医科大学药学院

出处《广西医科大学学报》 CAS 2015年第6期863-867,共5页 Journal of Guangxi Medical University

基金国家自然科学基金资助项目(No.81373403) 广西研究生科研创新课题资助项目(No.YCSZ2015119)

关键词内含子源性27nt-miRNA 管腔形成内皮型一氧化氮合酶 intronic 27nt-miRNA tube formation endothelial nitric oxide synthase

分类号 R-33 [医药卫生]

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