中文摘要：

前期工作表明,内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）第4内含子中的27碱基（nucleotide,nt）重复序列是27-nt microRNA的来源,并对eNOS具有重要的调节作用.为进一步探讨该内含子源性27-nt microRNA参与调节eNOS表达的分子机制及其在内皮细胞增殖中的可能作用,通过构建27-ntmicroRNA高表达质粒,用脂质体将该质粒转染人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）,Westernblot和RT-PCR检测该细胞系中eNOS蛋白和mRNA表达情况以及胞核转录因子的表达改变,并观察HUVEC增殖的变化情况.结果发现：27-ntmicroRNA高表达能降低eNOSmRNA的水平和蛋白质表达;同时对转录因子Sp1、Ap1的蛋白质表达也产生了不同程度的抑制作用;转染后细胞的生长速度比未转染的细胞明显减慢,尤其转染了27-nt microRNA的双倍长度突变体（pEGP-mut-54nt-mi）质粒的HUVEC,其生长倍增时间比正常对照组明显延长达49.4%.结果表明,27-nt microRNA明显抑制eNOS蛋白及其mRNA表达,同时HUVEC增殖受到明显抑制,转录因子Sp1和Ap1在27-ntmicroRNA对eNOS的表达调节中起重要作用.实验提示,内含子源性microRNA与转录因子共同参与对内皮细胞增殖及其相关性基因的表达调节,可能是众多真核细胞中某些疾病相关性基因表达自我调节的重要机制之一.

英文摘要：

Previously it was reported that the 27-nucleotide（nt） repeats in intron 4 of endothelial nitric syanthase （eNOS） were the source of 27-nt microRNA, which play an important role in regulation of eNOS expression. In order to further study the molecular mechanisms in regulation of eNOS gene expression and endothelial cell proliferation by the 27-nt microRNA, it was constructed the 27-nt microRNA highly expression plasmid, which was transferred into HUVEC with lipofectamine. The level of eNOS protein and mRNA, as well as Sp1 and Ap-1, were measured by Western blot and RT-PCR, respectively. The proliferation of the HUVEC was analyzed by MTT. The results showed that the 27-nt microRNA can significantly decrease eNOS mRNA level by 94.6% （0.015 ± 0.006 vs 0.277 ± 0.012, P 0.01） and inhibit its protein expression by 94.9%（0.012 ± 0.005 vs 0.237 ± 0.010, P 0.01）; Sp1 and Ap1 protein were significantly decreased by 14.0% （0.860 ± 0.013 vs 1.000 ±0.015, P 0.05） and by 22.0% （0.780 ± 0.033 vs 1.000 ± 0.052, P 0.05）, compared with control, respectively. The growth rate of HUVEC treated with the 27-nt microRNA high expression was significantly decreased, particularly the inhibition in the cell lines transfected with double-length and mutant of the 27-nt microRNA plasmid, by which the doubling-time of growth was increased by 49.4% （29.22 ± 0.25 vs 19.55 ± 0.19, P 0.05）, compared with control. The data suggested that intronic 27-nt microRNA significantly inhibit the eNOS expression and the proliferation of HUVEC, by the time the expression of transcription factors Sp-1 and Ap-1 were altered, strongly suggesting that 27-nt microRNA and transcription factors cooperatively regulate the expression of related genes, consequently the proliferation of HUVEC. Data from the present study may serve as one model of the critical mechanisms through which the intronic microRNA and transcription factor synergisticly were involved in the auto-regulation of disease-related gene expression.