Zhang Wenyu, Yan Limei, Li Yumei, Chen Wei, Hu Nan, Wang Hui, Ou Hesheng
College of Pharmacy, Guangxi Medical University, No. 22, Shuangyong Road, Nanning, 530021, People's Republic of China.
Mol Cell Biochem. 2015 Jul;405(1-2):281-9. doi: 10.1007/s11010-015-2418-y. Epub 2015 Apr 29.
This study is to investigate the effect of miRNA-24 on endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. Constructed high expression miRNA-24 plasmids were introduced into human umbilical vein endothelial cells (HUVECs) by liposome. Cell numbers were counted by a Hemocytometer, and cell proliferation was detected by MTT assay. The expression levels of eNOS and specificity protein 1 (Sp1) at mRNA and protein levels were, respectively, examined by real-time PCR, immunohistochemistry, and Western blotting. Compared with the control group, endothelial cell proliferation in miRNA-24 group significantly decreased by 64.24 % (6.53 ± 0.11 vs 18.26 ± 0.45, P < 0.01). The expression of eNOS at mRNA and protein levels in miRNA-24 group decreased by 64.21 % (0.34 ± 0.01 vs 0.95 ± 0.02, P < 0.05) and 82.86 % (0.072 ± 0.06 vs 0.42 ± 0.06, P < 0.05), respectively. Meanwhile, the expression of Sp1 at mRNA and protein levels in miRNA-24 group decreased by 64.64 % (0.35 ± 0.01 vs 0.99 ± 0.03, P < 0.05) and 60.34 % (0.23 ± 0.05 vs 0.58 ± 0.07, P < 0.05), respectively. In anti-miRNA-24 group, endothelial cell proliferation decreased by 33.46 % compared with the control group (12.15 ± 0.21 vs 18.26 ± 0.45, P < 0.01), while it increased by 46.25 % compared with the miRNA-24 group (12.15 ± 0.21 vs 6.53 ± 0.11, P < 0.01). The expression of eNOS at mRNA and protein levels in anti-miRNA-24 group decreased by 44.21 % (0.53 ± 0.04 vs 0.95 ± 0.02, P < 0.05) and by 30.95 %(0.29 ± 0.05 vs 0.42 ± 0.06, P < 0.05) compared with the control group, while it increased by 35.84 % (0.53 ± 0.04 vs 0.34 ± 0.01, P < 0.05) and by 75.17 % (0.29 ± 0.05 vs 0.072 ± 0.06, P < 0.05) compared with miRNA-24 group. The expression of Sp1 at mRNA and protein levels in ant-miRNA-24 group decreased by 36.36 % (0.63 ± 0.04 vs 0.99 ± 0.03, P < 0.05) and by 22.41 % (0.45 ± 0.06 vs 0.58 ± 0.07, P < 0.05) compared with the control group, while it increased by 44.44 % (0.63 ± 0.04 vs 0.35 ± 0.01, P < 0.05) and by 48.88 % (0.45 ± 0.06 vs 0.23 ± 0.05, P < 0.05) compared with miRNA-24 group. HUVECs proliferation and eNOS expression are significantly inhibited by miRNA-24. Sp1, which is regulated by miRNA-24, might act as one of the important factors involved in eNOS gene expression.
本研究旨在探讨miRNA - 24对内皮型一氧化氮合酶（eNOS）表达及血管内皮细胞增殖的影响。通过脂质体将构建的高表达miRNA - 24质粒导入人脐静脉内皮细胞（HUVECs）。用血细胞计数板计数细胞数量，采用MTT法检测细胞增殖。分别通过实时PCR、免疫组织化学和蛋白质印迹法检测eNOS和特异性蛋白1（Sp1）在mRNA和蛋白质水平的表达。与对照组相比，miRNA - 24组内皮细胞增殖显著降低64.24%（6.53±0.11对18.26±0.45，P<0.01）。miRNA - 24组eNOS在mRNA和蛋白质水平的表达分别降低64.21%（0.34±0.01对0.95±0.02，P<0.05）和82.86%（0.072±0.06对0.42±0.06，P<0.05）。同时，miRNA - 24组Sp1在mRNA和蛋白质水平的表达分别降低64.64%（0.35±0.01对0.99±0.03，P<0.05）和60.34%（0.23±0.05对0.58±0.07，P<0.05）。在抗miRNA - 24组中，内皮细胞增殖较对照组降低33.46%（12.15±0.21对18.26±0.45，P<0.01），而较miRNA - 24组增加46.25%（12.15±0.21对6.53±0.11，P<0.01）。抗miRNA - 24组eNOS在mRNA和蛋白质水平的表达较对照组分别降低44.21%（0.53±0.04对0.95±0.02，P<0.05）和30.95%（0.29±0.05对0.42±0.06，P<0.05），而较miRNA - 24组分别增加35.84%（0.53±0.04对0.34±0.01，P<0.05）和75.17%（0.29±0.05对0.072±0.06，P<0.05）。抗miRNA - 24组Sp1在mRNA和蛋白质水平的表达较对照组分别降低36.36%（0.63±0.04对0.99±0.03，P<0.05）和22.41%（0.45±0.06对0.58±0.07，P<0.05），而较miRNA - 24组分别增加44.44%（0.63±0.04对0.35±0.01，P<0.05）和48.88%（0.45±0.06对0.23±0.05，P<0.05）。miRNA - 24显著抑制HUVECs增殖和eNOS表达。受miRNA - 24调控的Sp1可能是参与eNOS基因表达的重要因素之一。
