Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-beta1 gene.
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-beta1 (hTGF-beta1) 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 isothiocyanate-labeled 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-beta1 determined by in vitro transcription experiments and luciferase assay. In cultured human VSMCs, Py-Im polyamide targeting hTGF-beta1 promoter significantly inhibited expressions of hTGF-beta1 mRNA and protein. These results indicate that the synthetic Py-Im polyamide designed to bind hTGF-beta1 promoter inhibited hTGF-beta1 gene and protein expression successfully. This novel agent will be used for the TGF-beta-related diseases as a gene therapy.
Lai
Yu-Mu
YM
Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.
Fukuda
Noboru
N
Ueno
Takahiro
T
Matsuda
Hiroyuki
H
Saito
Satoshi
S
Matsumoto
Koichi
K
Ayame
Hirohito
H
Bando
Toshikazu
T
Sugiyama
Hiroshi
H
Mugishima
Hideo
H
Serie
Kazuo
K
D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007202:Indicators and Reagents; D009757:Nylons / Q000138:chemical synthesis / Q000494:pharmacology*; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011758:Pyrroles / Q000138:chemical synthesis / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics*; D053773:Transforming Growth Factor beta1; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics
D007202:Indicators and Reagents; D009757:Nylons; D009841:Oligonucleotides; D011758:Pyrroles; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014404:Tubulin; D004247:DNA; C064028:endodeoxyribonuclease FseI; D015252:Deoxyribonucleases, Type II Site-Specific