Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-β1 gene
Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-β1 gene
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DOI：
10.1124/jpet.105.089086
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发表时间：
2005-11-01
影响因子：
3.5
通讯作者：
Serie, K
中科院分区：
文献类型：
--
作者：
Lai, YM;Fukuda, N;Serie, K
吡咯-咪唑（Py-Im）聚酰胺能与双螺旋DNA小槽内的预定碱基对结合，具有较高的亲和力。这些合成的小分子可以干扰转录因子与dna的相互作用，抑制或激活相应基因的转录。在本研究中，我们设计并合成了一种Py-Im聚酰胺，靶向脂肪特异性元件2 （FSE2）附近的人转化生长因子- β 1 （htgf - β 1）启动子的-545 ~ -539碱基对，抑制该基因的表达。凝胶迁移率转移实验表明，合成的Py-Im聚酰胺与相应的双链寡核苷酸结合，而错配聚酰胺不结合。培养2 ~ 48h后，在人血管平滑肌细胞（VSMCs）细胞核中检测到荧光素异硫氰酸酯标记的Py-Im聚酰胺。通过体外转录实验和荧光素酶测定，Py-Im聚酰胺显著降低htgf - β 1启动子活性。在培养的人VSMCs中，靶向htgf - β 1启动子的Py-Im聚酰胺显著抑制htgf - β 1 mRNA和蛋白的表达。这些结果表明，设计结合htgf - β 1启动子的合成Py-Im聚酰胺成功抑制了htgf - β 1基因和蛋白的表达。这种新型药物将作为一种基因疗法用于tgf - β相关疾病。
Pyrrole-imidazole (Py-Im) polyamides can bind to the predetermined base pairs in the minor groove of double-helical DNA with high affinity. These synthetic small molecules can interfere with transcription factor-DNA interaction and inhibit or activate the transcription of corresponding genes. In the present study, we designed and synthesized a Py-Im polyamide to target -545 to -539 base pairs of human transforming growth factor- beta 1 (hTGF-beta 1) promoter adjacent to the fat-specific element 2 (FSE2) to inhibit the expression of the gene. Gel mobility shift assay showed that the synthetic Py-Im polyamide binds to its corresponding double-strand oligonucleotides, whereas the mismatch polyamides did not bind. Fluorescein isothiocyanatelabeled Py-Im polyamide was detected in the nuclei of human vascular smooth muscle cells (VSMCs) after 2- to 48-h incubation. Py-Im polyamide significantly decreased the promoter activity of hTGF-beta 1 determined by in vitro transcription experiments and luciferase assay. In cultured human VSMCs, Py-Im polyamide targeting hTGF-beta 1 promoter significantly inhibited expressions of hTGF-beta 1 mRNA and protein. These results indicate that the synthetic Py-Im polyamide designed to bind hTGF-beta 1 promoter inhibited hTGF-beta 1 gene and protein expression successfully. This novel agent will be used for the TGF-beta-related diseases as a gene therapy.